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.     

Assessment of the effects of cinnamon leaf oil on rumen microbial fermentation using two continuous culture systems

Two continuous culture (CC) systems, the rumen simulation technique (Rusitec) and a dual-flow (DF) fermenter, were used to evaluate effects of the essential oil from cinnamon leaf (CIN) on rumen microbial fermentation. Incubations (d 1 through 8 for adaptation and d 9 through 16 for sampling) were conducted concurrently in the 2 systems, with CIN added at 0 (control) and 500 mg/L of rumen fluid culture. Eight Rusitec (920 mL; dilution rate = 2.9%/h) and 6 DF (1,300 mL; dilution rate = 6.3%/h) fermenters were randomly assigned to treatment. Inoculum was prepared from 4 ruminally cannulated lactating Holstein cows fed a total mixed ration consisting of 51% forage and 49% concentrate (dry matter basis). Ruminal pH, total volatile fatty acid (VFA) concentration, and diet digestibility were reduced by CIN addition in the Rusitec but were not affected by CIN administration in the DF. The addition of CIN in the Rusitec decreased apparent N disappearance, NH₃-N concentration, and molar proportions of branched-chain VFA. In contrast, in the DF no effect of CIN was observed on apparent N degradation, NH₃-N concentration, and molar proportion of branched-chain VFA. In the Rusitec, the molar proportion of acetate was similar between treatments on d 9 and 13, but was lower from d 10 to 12 and higher on d 14 to 16 with CIN than with control (interaction of treatment × sampling day). The molar proportion of acetate remained unaffected by CIN addition in the DF. In both CC systems, the molar proportion of propionate was decreased whereas that of butyrate was increased by CIN addition. In the DF, CIN decreased microbial N flow and efficiency of microbial protein synthesis. Protozoa numbers were lower with CIN than with control in both CC fermenters. In the Rusitec, CIN increased ¹⁵N enrichment in total bacterial fractions, but no effect was observed on the production of microbial N. This study showed that CIN exhibited antimicrobial activity in both CC systems, but the effects were more pronounced in the Rusitec than in the DF system. These differences are likely a reflection of the higher dilution rate in the DF resulting in a lower effective concentration of CIN than in Rusitec. Based on these changes in rumen microbial fermentation, supplementation of CIN at the concentration evaluated in this study may not be nutritionally beneficial to ruminants.     

Effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and nutrient utilization in dairy does

The objective of this study was to evaluate effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and on feed intake and nutrient utilization in dairy does. Batch cultures of mixed rumen microorganisms were used to study effects of different concentrations of fumarate on fermentation with various N sources (ammonia as ammonium bicarbonate, casein amino acids, casein peptides, gelatin peptides) and feeds (bermudagrass hay, mixed diet of 60% bermudagrass hay plus 40% concentrate) for 6 and 24 h, respectively. Substrates were grouped into pairs for separate incubations. Monosodium fumarate was added to incubation tubes to achieve final concentrations of 0, 5, and 10 mM fumarate. More ammonia accumulated at the end of incubation with added ammonium bicarbonate. Ammonia concentration was higher for peptide compared with amino acid incubation, and for casein peptide compared with gelatin peptide. Addition of fumarate linearly decreased ammonia for all N sources and for feed substrates. For all substrate types, fumarate treatment increased acetate, propionate, and total volatile fatty acids (VFA), decreased acetate to propionate ratio, and tended to reduce branched-chain VFA. Digestion of feed neutral detergent fiber (NDF) by rumen microorganisms was improved by fumarate along with elevated endoglucanase and xylanase activities. In an animal metabolism experiment, 8 dairy does (4 per treatment) were used in a completely randomized design for 21 d. Does were fed a hay plus concentrate diet without (control) or with fumarate (6 g/head per day) supplementation to determine feed intake, whole-tract nutrient digestibility, and N utilization. Fumarate treatment did not affect weight change or feed intake but increased whole-tract digestion of gross energy, crude protein, and cellulose. Digested N was increased by fumarate supplementation; however, N retention was unaffected. Plasma glucose concentration was elevated with fumarate but urea N concentration remained unchanged. Fumarate addition had significant effects on rumen microbial fermentation by decreasing ammonia and branched-chain VFA, and by increasing acetate and propionate, and NDF digestion. These effects were reflected in the improvement in whole-tract gross energy, crude protein, and cellulose digestion and elevated plasma glucose concentration when dairy does were supplemented with fumarate.     

Plant extracts affect in vitro rumen microbial fermentation

Different doses of 12 plant extracts and 6 secondary plant metabolites were incubated for 24 h in diluted ruminal fluid with a 50:50 forage:concentrate diet. Treatments were: control (no additive), plant extracts (anise oil, cade oil, capsicum oil, cinnamon oil, clove bud oil, dill oil, fenugreek, garlic oil, ginger oil, oregano oil, tea tree oil, and yucca), and secondary plant metabolites (anethol, benzyl salicylate, carvacrol, carvone, cinnamaldehyde, and eugenol). Each treatment was supplied at 3, 30, 300, and 3,000 mg/L of culture fluid. At 3,000 mg/L, most treatments decreased total volatile fatty acid concentration, but cade oil, capsicum oil, dill oil, fenugreek, ginger oil, and yucca had no effect. Different doses of anethol, anise oil, carvone, and tea tree oil decreased the proportion of acetate and propionate, which suggests that these compounds may not be nutritionally beneficial to dairy cattle. Garlic oil (300 and 3,000 mg/L) and benzyl salicylate (300 and 3,000 mg/L) reduced acetate and increased propionate and butyrate proportions, suggesting that methane production was inhibited. At 3,000 mg/L, capsicum oil, carvacrol, carvone, cinnamaldehyde, cinnamon oil, clove bud oil, eugenol, fenugreek, and oregano oil resulted in a 30 to 50% reduction in ammonia N concentration. Careful selection and combination of these extracts may allow the manipulation of rumen microbial fermentation.     

Effects of 3-nitrooxypropanol on methane emission,digestion, and energy and nitrogen balance of lactating dairy cows

The objective was to measure effects of 3-nitrooxypropanol (3NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and N metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and N balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3NP. Similarly, the decrease in N digestibility at the higher dose of the product was associated with a decrease in body N balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3NP. Twice-daily rumen dosing of 3NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product.     

Effects of patterns of suboptimal pH on rumen fermentation in a dual-flow continuous culture system

Low ruminal pH affects rumen fermentation, with the effects being larger as the time at suboptimal pH increases. Eight 1,325-mL dual-flow continuous culture fermenters were used to examine the hypothesis that the negative effects of a single cycle of 12 h (experiment 1) or 8 h (experiment 2) at pH 5.5 can be reduced by splitting it into several cycles. Temperature (39°C), diet (97 g/d of a 60:40 forage:concentrate diet), and solid (5%/h) and liquid (10%/h) dilution rates were kept constant. In experiment 1, treatments were a constant pH 6.4 (H); 1 cycle of 12 h at pH 5.5 (L12); 2 cycles of 6 h at pH 5.5; and 3 cycles of 4 h at pH 5.5. In experiment 2, treatments were a constant pH 6.4 (H); 1 cycle of 4 h at pH 5.5 (L4); 1 cycle of 8 h at pH 5.5 (L8); or 2 cycles of 4 h at pH 5.5. During the rest of the day, pH was maintained at 6.4. Each experiment consisted of 2 replicated periods of 8 d (5 d for adaptation and 3 d for sampling). Within period, treatments were randomly assigned to fermenters. Data were analyzed as a randomized complete block using PROC MIXED of SAS and differences declared at P < 0.05 using the Tukey's test. In experiment 1, L12 reduced neutral detergent fiber (NDF) digestion, acetate proportion, and the acetate:propionate ratio, increased propionate proportion, and tended to reduce ammonia N concentration, compared with H, but had no effect on the flow of dietary or microbial N, crude protein degradation, efficiency of microbial protein synthesis, or the flow of total, essential, and individual amino acids. Dividing the 12 h at suboptimal pH into 2 or 3 cycles reduced true organic matter (OM) degradation compared with H, and did not alleviate the negative effects on NDF digestion and volatile fatty acid profile observed in L12. In experiment 2, L4 tended to reduce true OM digestion, ammonia N concentration, and bacterial N flow, reduced CP degradation, and increased dietary N flow. Treatment L8 reduced OM and NDF digestion, and ammonia N concentration, compared with H. Treatments L4 and L8 also reduced acetate proportion and the acetate:propionate ratio, and increased propionate proportion and the flow of total, essential, and most individual amino acids, but had no effect on efficiency of microbial protein synthesis compared with the H treatment. When the 8 h at suboptimal pH was divided into 2 cycles of 4 h the effects were not different from L8. Results suggest that the effects of low pH are dependent on the total amount of time that pH is suboptimal and are not reduced by splitting it into various cycles.     

Effect of dietary nitrogen content and intravenous urea infusion on ruminal and portal-drained visceral extraction of arterial urea in lactating Holstein cows

Urea extraction across ruminal and portal-drained visceral (PDV) tissues were investigated using 9 rumen-cannulated and multi-catheterized lactating dairy cows adapted to low-N (12.9% crude protein) and high-N (17.1% crude protein) diets in a crossover design. The interaction between adaptation to dietary treatments and blood plasma concentrations of urea was studied by dividing samplings into a 2.5-h period without urea infusion followed by a 2.5-h period with primed continuous intravenous infusion of urea (0.493 ± 0.012 mmol/kg of BW per h). Cows were sampled at 66 ± 14 and 68 ± 12 d in milk and produced 42 ± 1 and 36 ± 1 kg of milk/d with the high-N and low-N diets, respectively. The arterial blood urea concentration before urea infusion was 1.37 and 4.09 ± 0.18 mmol/L with low-N and high-N, respectively. Dietary treatment did not affect the urea infusion-induced increase in arterial urea concentration (1.91 ± 0.13 mmol/L). Arterial urea extraction across the PDV and rumen increased from 2.7 to 5.4 ± 0.5% and from 7.1 to 23.8 ± 2.1% when cows were changed from high-N to low-N, respectively. Urea infusion did not decrease urea extractions, implying that urea transport rates were proportional to arterial urea concentrations. Urea extraction increased more across the rumen wall than across the total PDV for low-N compared with high-N, which implies that a larger proportion of total PDV uptake of arterial urea is directed toward the rumen with decreasing N intake. The ruminal vein - arterial (RA) concentration difference for ammonia increased instantly (first sampling 15 min after initiation of infusion) to the primed intravenous infusion when cows were adapted to the low-N diet. The RA difference for ammonia correlated poorly to the ventral ruminal concentration of ammonia (r = 0.55). Relating the RA difference for ammonia to a function of both ruminal ammonia concentration and the RA difference for urea markedly improved the fit (r = 0.85), indicating that a large fraction of ammonia released to the ruminal vein is absorbed from an epithelial ammonia pool not in equilibrium with the ventral ruminal ammonia pool. Changing cows from high-N to low-N affected the relative blood urea clearance by kidneys and PDV. The clearance by the kidneys decreased from 41 to 27 ± 2 L/h and the clearance by the PDV increased from 52 to 105 ± 12 L/h when the diet was changed from high-N to low-N. In conclusion, urea transport across gut epithelia in cattle is adapting to N status and driven by mass action. Data are commensurable with a model for urea transport across gut epithelia based on regulated expression or activity of facilitative urea transporters.     

Effects of ruminal ammonia and butyrate concentrations on reticuloruminal epithelial blood flow and volatile fatty acid absorption kinetics under washed reticulorumen conditions in lactating dairy cows

The effect of reticuloruminal epithelial blood flow on the absorption of propionate as a volatile fatty acid (VFA) marker in 8 lactating Holstein cows was studied under washed rumen conditions. The cows were surgically prepared with ruminal cannulas and permanent catheters in an artery and mesenteric, right ruminal, and hepatic portal veins. The experiment was designed with 2 groups of cows: 4 cows adapted to high crude protein (CP) and 4 to low CP. All cows were subjected to 3 buffers: butyric, ammonia, and control in a randomized replicated 3 × 3 incomplete Latin square design. The buffers (30 kg) were maintained in a temporarily emptied and washed rumen for 40 min. The initial concentration of VFA was 84.2 mmol/L. Butyrate was increased from 4 to 36 mmol/L in butyric buffer by replacement of acetate, and ammonia (NH₃) was increased from 2.5 to 22.5 mmol/L in ammonia buffer by replacement of NaCl. Increasing amounts of deuterium oxide (D₂O) were added to the buffers as the order of buffer sequence increased (6, 12, and 18 g of D₂O). Ruminal clearance of D₂O was used to estimate epithelial blood flow. To increase accuracy of the epithelial blood flow estimates, data of ruminal liquid marker (Cr-EDTA), and initial and final buffer volumes were fitted to a dynamic simulation model. The model was used to estimate ruminal liquid passages, residual liquid, and water influx (saliva and epithelia water) for each combination of cow and buffer (n = 24). Epithelial blood flow increased 49 ± 11% for butyric buffer compared with control. The ruminal disappearance of propionate (marker VFA) was affected by buffer and followed the same pattern as for epithelial blood flow. The correlation between ruminal disappearance of propionate and epithelial blood flow (r = 0.56) indicates that the removal of propionate can be limited by epithelial blood flow. The ruminal disappearance of propionate increased 30 ± 12% for the butyric compared with ammonia buffer and 12.5 ± 8% when compared with control. The net portal flux of propionate increased 32 ± 6% in butyric compared with control. In conclusion, rumen epithelial blood flow is positively correlated with ruminal disappearance of propionate and affects the kinetics of ruminal VFA absorption.     

Short communication: Comparison of pH,volatile fatty acids,and microbiome of rumen samples from preweaned calves obtained via cannula or stomach tube

The objective of this study was to compare rumen samples from young dairy calves obtained via a stomach tube (ST) or a ruminal cannula (RC). Five male Holstein calves (46 ± 4.0 kg of body weight and 11 ± 4.9 d of age) were ruminally cannulated at 15 d of age. Calves received 4 L/d of a commercial milk replacer (25% crude protein and 19.2% fat) at 12.5% dry matter, and were provided concentrate and chopped oats hay ad libitum throughout the study (56 d). In total, 29 paired rumen samples were obtained weekly throughout the study in most of the calves by each extraction method. These samples were used to determine pH and volatile fatty acids (VFA) concentration, and to quantify Prevotella ruminicola and Streptococcus bovis by quantitative PCR. Furthermore, a denaturing gradient gel electrophoresis was performed on rumen samples harvested during wk 8 of the study to determine the degree of similarity between rumen bacteria communities. Rumen pH was 0.30 units greater in ST compared with RC samples. Furthermore, total VFA concentrations were greater in RC than in ST samples. However, when analyzing the proportion of each VFA by ANOVA, no differences were found between the sampling methods. The quantification of S. bovis and P. ruminicola was similar in both extraction methods, and values obtained using different methods were highly correlated (R² = 0.89 and 0.98 for S. bovis and P. ruminicola, respectively). Fingerprinting analysis showed similar bacteria band profiles between samples obtained from the same calves using different extraction methods. In conclusion, when comparing rumen parameters obtained using different sampling techniques, it is recommended that VFA profiles be used rather than total VFA concentrations, as total VFA concentrations are more affected by the method of collection. Furthermore, although comparisons of pH across studies should be avoided when samples are not obtained using the same sampling method, the comparison of fingerprinting of a bacteria community or a specific rumen bacterium is valid.     

Pre- and postweaning attributes in faunated and ciliate-free calves fed calf starter with or without fish meal

In a 2 × 2 factorial design, 24 newborn, crossbred (Bos indicus × Bos taurus) calves were distributed in 4 equal groups involving dietary treatments of prestarter diets with (FM) or without fish meal (NFM) in a faunated (F) or ciliate-free (D) ruminal environment to study the ruminal fermentative development in pre-and postweaning periods. Defaunation was achieved by rearing calves in isolation and its effect was studied after first appearance of ciliate protozoa (observed after 8 wk of age) in the faunated animals. Calves were fed colostrum for 24 h and whole milk until weaning at 8 wk of age. Ruminal content samples were collected on d 4, 1 wk, weekly to 8 wk, and then biweekly at 9, 11, and 13 wk of age. The samples were analyzed for fermentation products [pH, total volatile fatty acids (VFA) and ammonia N] and enzyme [carboxymethyl (CM) cellulase, xylanase, β-glucosidase, α-amylase, β-galactosidase, proteases, and urease] activities. Weekly feed intake increased with age, but was similar in both groups. Ruminal pH declined steadily during 0 to 4 wk of age and then stabilized. The total VFA concentration increased with the age. The ammonia N (mg/dL) concentration increased from 14.9 on d 4 to 32.4 at 4 wk, decreased to 17.6 at 8 wk, and then steadied during the postweaning period. Samples collected on d 4 had no fibrolytic activity. Xylanase (U/dL) appeared first (1 wk) followed by β-glucosidase (U/dL) and CM cellulase (U/dL), which increased steadily from a low of 4.69, 0.08, and 2.95 to 31.8 (6 wk), 5.92 (7 wk), and 19.8 (8 wk), respectively, and the concentrations showed nonsignificant alterations during postweaning periods. The concentration of α-amylase (U/dL) increased from 34.3 on d 4 to 87.2 at 8 wk, and then decreased to 56.6 (13 wk). β-Galactosidase increased up to 6 wk then decreased to trace level (0.20 U/dL) at 13 wk of age. The concentrations of proteases and urease reached a steady state after 1 wk of age. The effect of diet type on ruminal fermentation products and enzyme parameters was nonsignificant. However, a steady and proportional alteration in both parameters in response to dry feed intake with the advancement of age was seen in all calves. Defaunation increased total VFA (97.3 vs. 75.8 mM/L) and α-amylase activity (80.3 vs. 61.4 U/dL) and decreased ammonia N (16.4 vs. 21.1 mg/dL), whereas the effect on other parameters was nonsignificant. Ruminal fermentative changes responded to dry feed intake, but did not differ in response to animal protein in prestarter diet.     

Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal fermentation,microbial abundance,and methane emissions in sheep

The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy propionate (E3NP) and 3-nitrooxypropanol (3NP), 2 recently developed compounds with potential antimethanogenic activity, in vitro and in vivo in nonlactating sheep on ruminal methane production, fermentation pattern, the abundance of major microbial groups, and feed degradability. Three experiments were conducted, 1 in vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2 doses of E3NP and 3NP (40 and 80 μL/L), which showed a substantial reduction of methane production (up to 95%) without affecting concentration of volatile fatty acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30 d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats (60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After 7, 14, and 15 d of treatment, methane emissions were recorded in respiration chambers and rumen fluid samples were collected for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. Methane production decreased by 29% compared with the control with the higher dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was observed without detrimental effects on dry matter intake. In experiment 3, 9 adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay and oats (60:40), were treated with E3NP or 3NP at one dose (100 mg/animal per day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA analysis and quantification of bacterial, protozoal, and archaeal numbers by real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were incubated in the rumen of sheep to determine dry matter ruminal degradation over 24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3NP groups, respectively). A reduction in methane production was observed for both additives at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was significantly decreased. Likewise, total concentrations of the analyzed microbial groups in the rumen showed no difference among treatments and doses for both experiments. Both tested compounds showed promise as methane inhibitors in the rumen, with no detrimental effects on fermentation or intake, which would need to be confirmed in lactating animals.     

Effect of garlic oil and four of its compounds on rumen microbial fermentation

Different concentrations (3, 30, 300, and 3000 mg/L of culture fluid) of garlic oil (GAR), diallyl sulfide (DAS), diallyl disulfide (DAD), allicin (ALL), and allyl mercaptan (ALM) were incubated for 24 h in diluted ruminal fluid with a 50:50 forage:concentrate diet (17.7% crude protein; 30.7% neutral detergent fiber) to evaluate their effects on rumen microbial fermentation. Garlic oil (30 and 300 mg/L), DAD (30 and 300 mg/L), and ALM (300 mg/L) resulted in lower molar proportion of acetate and higher proportions of propionate and butyrate. In contrast, at 300 mg/L, DAS only increased the proportion of butyrate, and ALL had no effects on volatile fatty acid proportions. In a dual-flow continuous culture of rumen fluid fed the same 50:50 forage:concentrate diet, addition of GAR (312 mg/L), DAD (31.2 and 312 mg/L), and ALM (31.2 and 312 mg/L) resulted in similar changes to those observed in batch culture, with the exception of the lack of effect of DAD on the proportion of propionate. In a third in vitro study, the potential of GAR (300 mg/L), DAD (300 mg/L), and ALM (300 mg/L) to decrease methane production was evaluated. Treatments GAR, DAD, and ALM resulted in a decrease in methane production of 73.6, 68.5, and 19.5%, respectively, compared with the control. These results confirm the ability of GAR, DAD, and ALM to decrease methane production, which may help to improve the efficiency of energy use in the rumen.     

Effects of essential oils on rumen fermentation, milk production, and feeding behavior in lactating dairy cows

Seven ruminally cannulated lactating Holstein dairy cows were used in an incomplete Latin rectangle design to assess the effects of 2 commercial essential oil (EO) products on rumen fermentation, milk production, and feeding behavior. Cows were fed a total mixed ration with a 42:58 forage:concentrate ratio (DM basis). Treatments included addition of 0.5 g/d of CE Lo (85 mg of cinnamaldehyde and 140 mg of eugenol), 10 g/d of CE Hi (1,700 mg of cinnamaldehyde and 2,800 mg of eugenol), 0.25 g/d of CAP (50 mg of capsicum), or no oil (CON). Cows were fed ad libitum twice daily for 21 d per period. Dry matter intake, number of meals/d, h eating/d, mean meal length, rumination events/d, h ruminating/d, and mean rumination length were not affected by EO. However, length of the first meal after feeding decreased with addition of CE Hi (47.2 min) and CAP (49.4 min) compared with CON (65.4 min). Total volatile fatty acids, individual volatile fatty acids, acetate:propionate ratio, and ammonia concentration were not affected by EO. Mean rumen pH as well as bouts, total h, mean bout length, total area, and mean bout area under pH 5.6 did not differ among treatments. Total tract digestibility of organic matter, dry matter, neutral detergent fiber, acid detergent fiber, crude protein, and starch were not affected by EO. Milk yield and composition did not change with EO. In situ dry matter disappearance of ground soybean hulls was not affected by EO. However, organic matter disappearance of soybean hulls with CE Hi tended to decrease compared with CON. Compared with CON, neutral detergent fiber disappearance (41.5 vs. 37.6%) and acid detergent fiber disappearance (44.5 vs. 38.8%) decreased with addition of CE Hi. The CE Lo had no effect on rumen fermentation, milk production, or feeding behavior but CAP shortened the length of the first meal without changing rumen fermentation or production, making it a possible additive for altering feeding behavior. The CE Hi negatively affected rumen fermentation and shortened the length of the first meal, suggesting that a dose of 10 g/d is not beneficial to lactating dairy cows.     

Diet supplementation with thyme oil and its main component thymol failed to favorably alter rumen fermentation,improve nutrient utilization,or enhance milk production in dairy cows

Phenolic compounds and essential oils with high content of phenolic compounds have been reported to exert antimicrobial activities in vitro. The objective of this study was to determine the effects of dairy cow diet supplementation with thyme oil and its main component thymol on intake and total-tract apparent digestibility of nutrients, rumen fermentation characteristics, ruminal protozoa, nitrogen excretion, and milk production. For this aim, we used 8 multiparous, ruminally cannulated Holstein cows in a replicated 4 × 4 Latin square design (28 d periods), balanced for residual effects. Cows were fed 1 of the 4 following experimental treatments: total mixed ration (TMR) with no additive (control); TMR + monensin [24 mg/kg of dry matter (DM)]; TMR + thyme oil (50 mg/kg of DM); and TMR + thymol (50 mg/kg of DM). Compared with the control diet, feeding thyme oil or thymol had no effect on DM intake, nutrient total-tract apparent digestibility, total N excretion, ruminal pH, ammonia concentration, total volatile fatty acid (VFA) concentration, or acetate:propionate ratio. Ruminal protozoa density was not modified by thyme oil, but decreased with thymol supplementation. Supplementation with thyme oil or thymol did not affect milk production, milk composition, or efficiency of milk production. Neither thyme oil nor thymol affected efficiency of dietary N use for milk N secretion (N intake/milk N). Supplementation with monensin tended to decrease DM intake (–1.2 kg/d) and milk fat yield. Total-tract apparent digestibility of nutrients did not differ between cows fed monensin and cows fed the control diet. Total VFA concentration was not changed by monensin supplementation compared with control, but adding monensin shifted the VFA profile toward more propionate and less acetate, resulting in a decrease of acetate:propionate ratio. Protozoa density and ammonia concentration were lower in the ruminal content of cows fed monensin compared with that of cows fed the control diet. Total N excretion was not affected by monensin supplementation. Likewise, efficiency of use of dietary N for milk N secretion was unchanged in cows fed monensin. The results of this study contrasted with the claimed in vitro antimicrobial activity of thyme oil and thymol: we observed no positive effects on rumen metabolism (i.e., N and VFA) or milk performance in dairy cows. Under the conditions of this study, including thyme oil or thymol at 50 mg/kg of DM had no benefits for rumen fermentation, nutrient utilization and milk performance in dairy cows.     

Nutrient utilization of differing forage-to-concentrate ratios by growing Holstein heifers

Traditionally, high-forage, low-concentrate diets fed ad libitum have been the primary system of feeding dairy heifers. However, high-concentrate diets can be fed at restricted intakes to reach desired rates of gain and increase nutrient efficiency. A total mixed ration containing high corn silage (CS; HCS: 77% CS, 23% concentrate) or low CS (LCS: 67% concentrate, 33% CS) was fed at restricted intakes in 2 trials to evaluate nutrient utilization by growing heifers. In the first trial, 4 ruminally cannulated heifers (298 ± 16 kg of body weight) were fed to study differences in rumen pH, volatile fatty acid and ammonia concentrations, and mass of rumen contents. In situ determinations were made on the total mixed ration and CS. Low CS rations were digested more rapidly in situ when compared with HCS (4.5 vs. 2.3 ± 0.3%/h), and no differences were observed in CS digestibility when incubated in the rumen of heifers fed either ration. Mean rumen pH tended to be lower for LCS than for HCS (5.9 vs. 6.2 ± 0.1). Individual and total rumen volatile fatty acid concentrations and rumen ammonia concentration were not different between treatments. Total mass of rumen contents was lower for LCS. In the second trial, four 6-mo-old heifers (172 ± 14 kg of body weight) and four 12-mo-old heifers (337 ± 10 kg of body weight) were used. Digestibility of dry matter was greater for the LCS than the HCS diet in both age groups (76.3 vs. 71.1% for 12-mo-old heifers; 71.4 vs. 68.9% for 6-mo-old heifers). Apparent digestibility of N was not different between treatments; however, retained N was higher for the LCS diets for both age groups. Fecal output was significantly reduced in the LCS diets for both age groups. Feeding low-forage, high-concentrate diets to growing dairy heifers at restricted intakes, although more highly digestible, resulted in few significant differences in rumen fermentation patterns and lower fecal output.     

Effect of dietary concentrate on rumen fermentation, digestibility, and nitrogen losses in dairy cows

The objective of this experiment was to investigate the effect of level of dietary concentrate on rumen fermentation, digestibility, and N losses in lactating dairy cows. The experiment was a replicated 3 × 3 Latin square design with 6 cows and 16-d adaptation periods. Ruminal contents were exchanged between cows at the beginning of each adaptation period. Data for 2 of the diets tested in this experiment are presented here. The diets contained (dry matter basis): 52% (LowC; control) and 72% (HighC) concentrate feeds. Crude protein contents of the diets were 16.5 and 16.4%, respectively. The HighC diet decreased ruminal pH and ammonia concentration and increased propionate concentration compared with LowC. Acetate:propionate ratio was greater for LowC than for HighC. Rumen methane production and microbial protein synthesis were unaffected by diet. Dry matter intake was similar among diets, but milk yield was increased by HighC compared with LowC (36.0 and 33.2 kg/d, respectively). Milk fat percentage and yield and total-tract apparent NDF digestibility were decreased by HighC compared with LowC. More ruminal ammonia N was transferred into milk protein with HighC than with LowC. Urinary N excretion, plasma urea N, and milk urea N concentration were not affected by diet. The ammonia emitting potential of manure was similar between LowC and HighC diets. Increased concentrate proportion in the diet of dairy cows resulted in reduced ruminal ammonia concentration and enhanced ammonia utilization for milk protein synthesis. These effects, however, did not result in reduced urinary N losses and only marginally improved milk N efficiency. Increasing dietary concentrate was not a successful strategy to mitigate enteric methane production and ammonia emissions from manure.     

Prediction of ammonia emission from dairy cattle manure based on milk urea nitrogen: Relation of milk urea nitrogen to ammonia emissions

The main objectives of this study were to assess the relationship between ammonia emissions from dairy cattle manure and milk urea N (MUN; mg/dL) and to test whether the relationship was affected by stage of lactation and the dietary crude protein (CP) concentration. Twelve lactating multiparous Holstein cows were randomly selected and blocked into 3 groups of 4 cows intended to represent early [123 ± 26 d in milk (DIM)], mid (175 ± 3 DIM), and late (221 ± 12 DIM) lactation stages. Cows within each stage of lactation were randomly assigned to a treatment sequence within a split-plot Latin square design balanced for carryover effects. Stage of lactation formed the main plots (squares) and dietary CP levels (15, 17, 19, and 21% of diet dry matter) formed the subplots. The experimental periods lasted 7 d, with d 1 to 6 used for adjustment to diets and d 7 used for total collection of feces and urine as well as milk sample collection. The feces and urine from each cow were mixed in the proportions in which they were excreted to make slurry that was used to measure ammonia emissions at 22.5°C over 24 h using flux chambers. Samples of manure slurry were taken before and after ammonia emission measurements. The amount of slurry increased by 22% as dietary CP concentration increased from 15 to 21%, largely because of a greater urine volume (25.3 to 37.1 kg/d). Initial urea N concentration increased linearly with dietary CP from 153.5 to 465.2 mg/dL in manure slurries from cows fed 15 to 21% CP diets. Despite the large initial differences, the final concentration of urea N in manure slurries was less than 10.86 mg/dL for all dietary treatments. The final total ammoniacal N concentration in manure slurries increased linearly from 228.2 to 508.7 mg/dL as dietary CP content increased from 15 to 21%. Ammonia emissions from manure slurries ranged between 57 and 149 g of N/d per cow and increased linearly with dietary CP content, but were unaffected by stage of lactation. Ammonia emission expressed as a proportion of N intake increased with percentage CP in the diet from about 12 to 20%, whereas ammonia emission as a proportion of urinary urea N excretion decreased from 67 to 47%. There was a strong relationship between ammonia emission and MUN [ammonia emission (g/d per cow) = 25.0 (±6.72) + 5.03 (±0.373) × MUN (mg/dL); R² = 0.85], which was not different among lactation stages. Milk urea N concentration is one of several factors that allows prediction of ammonia emissions from dairy cattle manure.     

Effects of dietary protein concentration and coconut oil supplementation on nitrogen utilization and production in dairy cows

The objective of this study was to investigate the effect of metabolizable protein (MP) deficiency and coconut oil supplementation on N utilization and production in lactating dairy cows. The hypothesis of the study was that a decrease in ruminal protozoal counts with coconut oil would increase microbial protein synthesis in the rumen, thus compensating for potential MP deficiency. The experiment was conducted for 10 wk with 36 cows (13 primiparous and 23 multiparous), including 6 ruminally cannulated cows. The experimental period, 6 wk, was preceded by 2-wk adaptation and 2-wk covariate periods. Cows were blocked by parity, days in milk, milk yield, and rumen cannulation and randomly assigned to one of the following diets: a diet with a positive MP balance (+44 g/d) and 16.7% dietary crude protein (CP) concentration (AMP); a diet deficient in MP (−156 g/d) and 14.8% CP concentration (DMP); or DMP supplemented with approximately 500 g of coconut oil/head per day (DMPCO). Ruminal ammonia tended to be greater and plasma urea N (20.1, 12.8, and 13.1 mg/dL, for AMP, DMP, and DMPCO diets, respectively) and milk urea N (12.5, 8.3, and 9.5 mg/dL, respectively) were greater for AMP compared with DMP and DMPCO. The DMPCO diet decreased total protozoa counts (by 60%) compared with DMP, but had no effect on the methanogens profile in the rumen. Total tract apparent digestibility of dry matter and CP was decreased by DMP compared with AMP. Fiber digestibility was lower for both DMP and DMPCO compared with AMP. Urinary N excretion was decreased (by 37%) by both DMP and DMPCO compared with AMP. The DMP and DMPCO diets resulted in greater milk N efficiency compared with AMP (32.0 and 35.1 vs. 27.6%, respectively). Milk yield was decreased by both DMP and DMPCO compared with AMP (36.2, 34.4, and 39.3 kg/d, respectively) and coconut oil supplementation suppressed feed intake and caused milk fat depression. Coconut oil supplementation decreased short-chain fatty acid (C4:0, C6:0, and C8:0) concentration and increased medium-chain (C12:0 and C14:0) and total trans fatty acids in milk. Overall, the MP-deficient diets decreased N losses, but could not sustain milk production in this study. Coconut oil decreased feed intake and similar to DMP, suppressed fiber digestibility. Despite decreased protozoal counts, coconut oil had no effect on the methanogen population in the rumen.     

Effect of abomasal pectin infusion on digestion and nitrogen balance in lactating dairy cows

Two experiments were conducted to test the hypothesis that increasing carbohydrate fermentation in the large intestine would increase intestinal conversion of blood urea N to microbial protein, thereby reducing urinary N output. In experiment 1, 3 multiparous Holstein cows were used in an incomplete 4 × 4 Latin square with 14-d periods. Cows were fed the same basal diet and treatments were the abomasal infusion of 0, 0.5, or 1 kg/d of citrus pectin, or the addition of 1 kg/d of molasses to the basal diet. Experiment 2 used 6 cows in a double reversal design with four 21-d periods. Cows were fed one basal diet and treatments were the abomasal infusion of either 0 or 1 kg/d of pectin. In experiment 1, pectin infusion linearly decreased basal ration intake from 25.0 to 23.2 kg/d. This was prevented in experiment 2 by restricted feeding, and basal ration intake was 22.2 kg/d. Abomasal pectin caused numeric decreases in total tract apparent digestibility of neutral detergent fiber and neutral detergent solubles in experiment 1 and significantly decreased starch digestibility in experiment 2, suggesting that pectin may have reduced postruminal nutrient digestibility. Pectin infusion did not affect milk yield but decreased milk fat percentage from 3.69 to 3.53% in experiment 2. Increasing abomasal pectin tended to decrease urinary N and increase fecal N in experiment 1 and these effects were significant in experiment 2. For both experiments, urinary N decreased 26 g/d, approximately 10% of daily urine N output. Abomasal pectin did not affect fecal pH or DM content; however, in experiment 2, pectin decreased fecal ammonia from 19.8 to 13.4 mmol/kg of DM and increased fecal purines from 13.8 to 15.8 mmol/kg of DM. In both experiments, excretion of fecal purines was increased from 15 g/d for 0 kg/d pectin to 18 g/d for 1 kg/d pectin, although this increase was only significant in experiment 2. These results suggest that manipulating dairy diets to increase postruminal fermentation may reduce urinary N and consequently manure ammonia losses. However, abomasal pectin tended to decrease both ruminal ammonia concentration and urinary purine derivative output in experiment 2, suggesting that postruminal pectin fermentation may have compromised rumen microbial protein production.     

Effects of low rumen-degradable protein or abomasal fructan infusion on diet digestibility and urinary nitrogen excretion in lactating dairy cows

Post-ileal carbohydrate fermentation in dairy cows converts blood urea nitrogen (BUN) into fecal microbial protein. This should reduce urinary N, increase fecal N, and reduce manure NH₃ volatilization. However, if intestinal BUN recycling competes with ruminal BUN recycling, hindgut fermentation may reduce NH₃ for rumen microbial protein synthesis. Eight lactating Holstein cows were used in a replicated 4 × 4 Latin square design with 14-d periods. Treatments were arranged as a 2 × 2 factorial. Diets contained either adequate rumen-degradable protein (RDP; high RDP) or were 28% below predicted RDP requirements (low RDP). Cows received abomasal infusions of either 10 L/d of saline or 10 L/d of saline containing 1 kg/d of inulin. We hypothesized that reducing ruminal NH₃, either by restricting RDP intake or by diverting BUN to feces with inulin, would reduce rumen microbial protein synthesis, as would be evidenced by significant main effects of treatments on rumen NH₃, milk production, and urinary purine derivative excretion. Furthermore, we thought it likely that effects of inulin might be greater when rumen NH₃ was already low, as would be indicated by significant interactions between inulin infusion and dietary RDP level on rumen NH₃, milk production, and urinary purine derivative excretion. Rumen NH₃ was reduced by the low-RDP diet, but urinary purine derivative excretion and milk production were unaffected. However, the low-RDP diet reduced apparent total tract digestibility of OM and starch and reduced in situ rumen NDF digestibility. Abomasal inulin reduced the BUN concentration but did not affect milk yield or rumen NH₃, suggesting that RDP requirements are not affected by hindgut fermentation. Inulin shifted 23 g/d of N from urine to feces. However, based on fecal purine excretion, we estimated that only 8 g/d of the increased fecal N was due to increased fecal microbial output. Inulin reduced true digestibility of dietary protein or increased nonmicrobial as well as microbial endogenous losses. This latter effect may be an artifact of our experimental model that delivers easily fermented, soluble fiber to the small intestine. Normal dietary alterations to similarly increase large intestinal fermentation would probably arise from larger quantities of less rapidly digested carbohydrates. Increasing hindgut fermentation in practical diets should reduce manure NH₃ volatilization without impairing rumen fermentation, but the reduction is likely to be small.