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A Blend of Essential Plant Oils Used as an Additive to Alter Silage Fermentation or Used as a Feed Additive for Lactating Dairy Cows

      Abstract

      A blend of essential plant oils was evaluated for its effects on silage fermentation and animal performance. In the first experiment, the blend of essential oils was mixed with freshly chopped whole-plant corn to achieve a concentration of 0, 40, or 80 mg of active product per kilogram of fresh forage weight. Whole-plant corn was also mixed with a buffered propionic acid-based product at 0.2% of fresh forage weight. The blend of essential oils did not affect the populations of yeasts, molds, lactic acid bacteria, or enterobacteria; the fermentation end products; or the aerobic stability of the corn silage. Addition of the buffered propionic acid additive moderately reduced the production of acids during fermentation and resulted in a small reduction in the numbers of yeasts after ensiling, but did not affect aerobic stability. In a second experiment, 30 Holstein cows (4 primiparous and 26 multiparous) averaging 118 ± 70 d in milk and producing 38 ± 16 kg of milk/d were fed a total mixed ration, once daily, that consisted of (on a DM basis) 25% corn silage, 15% alfalfa silage, 10% alfalfa hay, and 50% concentrate. One-half of the cows were fed a blend of essential oils that was mixed directly into their total mixed ration to provide 1.2 g/cow per d for 9 wk. Cows fed the essential oils ate 1.9 kg more dry matter/d and produced 2.7 kg more 3.5% fat-corrected milk/d than did cows fed the control diet. The percentages of milk fat and protein, the somatic cell count numbers, and the concentrations of milk urea nitrogen were unaffected by treatment. Feed efficiency, change in body weight, and change in body condition scoring were also similar between treatments. After 12 h of incubation, the addition of a moderate dose and a high dose of essential oils to in vitro ruminal fermentations had no effect on the concentration of total VFA compared with the control treatment. However, they decreased the molar proportions of acetic, butyric, and valeric acids and increased the proportion of propionic acid. The blend of essential oils evaluated in this study altered in vitro ruminal fermentation and improved animal performance when fed directly to cows, but it did not affect the fermentation or aerobic stability of corn silage.

      Key words

      Introduction

      Essential oils are aromatic oily liquids extracted from plant material via expression, fermentation, or, commonly, a distillation method. They have a wide range of antimicrobial activities (
      • Chao S.C.
      • Young D.G.
      • Oberg C.J.
      Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses.
      ;
      • Burt S.
      Essential oils: Their antibacterial properties and potential applications on foods—A review.
      ), and interest in their use has been heightened because of the current emphasis on the use of natural compounds. Essential oils have been shown to accumulate in cell membranes and disrupt their integrity, leading to leakage of enzymes and metabolites (
      • Smid E.J.
      • Gorris L.G.M.
      Natural antimicrobials for food preservation.
      ). Structure-function relationships associated with polar groups, number of double bonds, molecular size, and molecular solubility may have marked effects on their activities (
      • Kamel C.
      Tracing modes of action and the roles of plant extracts in nonruminants.
      ). Essential oils may have applications in ruminant nutrition because fermentations in the silo and rumen are dependent on microbial activities that may be affected by their use.
      Essential oils have been shown to affect several rumen processes (
      • Calsamiglia S.
      • Busquet M.
      • Cardozo P.W.
      • Castillejos L.
      • Ferret A.
      Essential oils as modifiers of rumen microbial fermentation.
      ), but the results have been inconsistent. For example, deaminase activity in the rumen was decreased by their addition (
      • Newbold C.J.
      • McIntosh F.M.
      • Williams P.
      • Losa R.
      • Wallace R.J.
      Effects of a specific blend of essential oil compounds on rumen fermentation.
      ;
      • Wallace R.J.
      Antimicrobial properties of plant secondary metabolites. Symposium: Plants as animal foods: A case of catch 22.
      ). Specifically,
      • McIntosh F.M.
      • Williams P.
      • Losa R.
      • Wallace R.J.
      • Beever D.A.
      • Newbold C.J.
      Effects of essential oils on ruminal microorganisms and their protein metabolism.
      observed that Peptostreptococcus anaerobius, Clostridium stricklandii, and Prevotella ruminicola, species known as “hyperammonia-producing” bacteria (
      • Russell J.B.
      • Onodera R.
      • Hino T.
      Ruminal protein fermentation: New perspectives on previous contradictions.
      ), were inhibited by a commercial blend of plant oils and suggested that the main effect of plant oils occurred during the final phase of protein degradation.
      • Ferme D.
      • Banjac M.
      • Calsamiglia S.
      • Busquet M.
      • Kamel C.
      • Avguštin G.
      The effect of plant extracts on microbial community structure in a rumen-simulating continuous-culture system as revealed by molecular profiling.
      also observed a reduction in bacteria from the proteolytic genus Prevotella in a ruminal continuous culture system when treated with a garlic extract. However,
      • Benchaar C.
      • Petit H.V.
      • Berthiaume R.
      • Ouellet D.R.
      • Chiquette J.
      • Chouinard P.Y.
      Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production, and milk composition in dairy cows.
      and
      • Castillejos L.
      • Calsamiglia S.
      • Ferret A.
      • Losa R.
      Effects of a specific blend of essential oil compounds and the type of diet on rumen microbial fermentation and nutrient flow from a continuous culture system.
      reported that a blend of essential oils had no effect on ruminal ammonia. Essential oils have also had varied effects on ruminal VFA patterns. Dried extracts of Lavandula officinalis stimulated in vitro rumen production of VFA (
      • Broudiscou L.P.
      • Lassalas B.
      Effects of Lavandula officinalis and Equisetum arvense dry extracts and isoquercitrin on the fermentation of diets varying in forage contents by rumen microorganisms in batch culture.
      ).
      • Castillejos L.
      • Calsamiglia S.
      • Ferret A.
      • Losa R.
      Effects of a specific blend of essential oil compounds and the type of diet on rumen microbial fermentation and nutrient flow from a continuous culture system.
      reported that a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) increased the acetate:propionate ratio in ruminal fermentations, but that effects may vary based on the diet and conditions in the rumen.
      • Cardozo P.W.
      • Calsamiglia S.
      • Ferret A.
      • Kamel C.
      Screening for the effects of a natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle.
      , using an in vitro system with ruminal fluid from steers fed a high-concentrate diet, reported that cinnamon oil and cinnamaldehyde increased the acetate:propionate ratio and decreased VFA concentration when the culture pH was 7. However, when the culture pH was 5.5, the acetate:propionate ratio decreased and total VFA increased.
      Although not directly studied to date, essential oils may have the potential to alter the ensiling of forage crops via their effects on microbes associated with this process.
      • Kivanc M.
      • Akgulm A.
      • Dogan A.
      Inhibitory and stimulatory effects of cumin, oregano and their essential oils on growth and acid production of Lactobacillus plantarum and Leuconostoc mesenteroides.
      reported that cumin (from Cuminum cyminum L.) stimulated growth and lactic acid production of Lactobacillus plantarum. This bacterium is a common component of inoculants added to stimulate silage fermentation (
      • Kung Jr, L.
      • Stokes M.A.
      • Lin C.J.
      Silage additives.
      ). Capsaicin, from peppers (Capsicum), has been shown to inhibit species of Clostridium (
      • Cichewicz R.H.
      • Thorpe P.A.
      The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine.
      ;
      • Kamel C.
      Tracing modes of action and the roles of plant extracts in nonruminants.
      ) that can dominate the fermentation process in wet silages and lead to extensive degradation of nutrients. In addition, clove and oregano extracts (containing eugenol) have been shown to be highly effective in inhibiting the growth of Clostridium botulinum (
      • Ismaiel A.
      • Pierson M.D.
      Inhibition of growth and germination of C. botulinum 33A, 40B, and 1623E by essential oil of species.
      ). Species of yeasts that are closely associated with aerobic spoilage in silages have also been inhibited by some essential oils. For example, carvacrol inhibited the growth of some strains of Saccharomyces cerevisiae (
      • Knowles J.
      • Roller S.
      Efficacy of chitosan, carvacrol, and a hydrogen peroxide-based biocide against foodborne microorganisms in suspension and adhered to stainless steel.
      ), and thymol has inhibited the growth of Debaryomyces hansenii (
      • Curtis O.F.
      • Hetty K.S.
      • Cassagnol G.
      • Peleg M.
      Comparison of the inhibitory and lethal effects of synthetic versions of plant metabolites (anethole, carvacrol, eugenol, and thymol) on food spoilage yeast (Debaromyces hansenii).
      ).
      • Juglal S.
      • Govinden R.
      • Odhav B.
      Spice oils for the control of co-occurring mycotoxin-producing fungi.
      reported antifungal activities of essential oils toward fungi-producing mycotoxins. To date, we know of no published studies evaluating the use of essential oils to alter the fermentation and aerobic stability of corn silage.
      The objectives of this study were to 1) determine the effect of a specific blend of essential oils on the fermentation and aerobic stability of corn silage, and 2) to determine its effects on DMI, milk production, and milk composition when fed directly to lactating cows.

      Materials and Methods

      Silage Experiment

      Corn forage was harvested at 28 to 29% of DM with a New Holland FP230 pull-type harvester (New Holland North America, New Holland, PA) with an on-board kernel processor. Chop length was set to achieve a theoretical cut of 0.95 cm. Four piles of chopped forage were treated with the following: 1) nothing, 2) a water-soluble formulation of essential oils (Crina, DSM Nutritional Products Ltd., 40% active ingredients) supplying 40 mg of active ingredients/kg of wet forage, 3) the same formulation supplying 80 mg of essential oils/kg of wet forage, or 4) 0.2% of a liquid buffered propionic acid-based product (Ultra Curb, Kemin Industries, West Des Moines, Iowa)/t of wet forage. The Crina product was a defined and patented blend of natural and natural-identical essential oil compounds that included thymol, eugenol, vanillin, and limonene on an organic carrier (
      • McIntosh F.M.
      • Williams P.
      • Losa R.
      • Wallace R.J.
      • Beever D.A.
      • Newbold C.J.
      Effects of essential oils on ruminal microorganisms and their protein metabolism.
      ). The low treatment was calculated hypothetically to supply the equivalent of 1,000 mg of the active essential oil blend for a cow consuming 25 kg of wet silage/d. All treatments were mixed in 250 mL of water and sprayed onto the chopped forage mass (50 kg of wet forage) while mixing. A similar amount of water was added to the control forage. Untreated and treated forages were packed into replicated mini silos (approximately 300 g capacity, 5 cm diameter × 20 cm height) and 4 silos were opened for each treatment after 3 and 8 d of ensiling. Approximately 14 kg of untreated and treated forages were also packed into 20-L macro silos (27 cm diameter × 36 cm height, 4 per treatment) and ensiled for 256 d. Packing densities in the silos were 217 ± 7.5 kg of DM/m3. Silos were stored in a room with temperatures between 20 and 26°C.
      Fresh forages and silage samples from each silo were evaluated for DM content by drying duplicate samples for 48 h in a forced-air oven set at 60°C. Water extracts were prepared from fresh and ensiled samples by mixing 25 g of forage with 225 mL of 25% Ringer's solution (Oxoid BR52, Basingstoke, UK) and homogenizing this mix for 1 min. The pH of the water extract was measured, and a portion of it was filtered through Whatman 54 filter paper (Whatman, Clifton, NJ) and acidified with 50% H2SO4 and frozen before analysis for ammonia-N by using a phenol-hypochlorite method (
      • Okuda H.
      • Fuji S.
      • Kawashima Y.
      A direct colorimetric method for blood ammonia.
      ). Lactic acid and VFA were analyzed on the filtered water extract as described by
      • Kung Jr, L.
      • Ranjit N.K.
      The effect of Lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage.
      . Water-soluble carbohydrates in forages and silages were determined by using the colorimetric method described by
      • Nelson N.
      A photometric adaptation of the Somogyi method for the determination of glucose.
      . A portion of the water extract was pour plated by using serial 10-fold dilutions on violet red bile glucose agar (Oxoid CM485) with a single overlay to determine the number of enterobacteria (d 0, 3, and 8 only) in samples. Plates were incubated under aerobic conditions at 36°C for 18 h. Yeast and molds (d 0, 3, 8, and 256) were determined by pour plating in malt extract agar (Oxoid CM59) that had been acidified (0.5% vol/vol) after autoclaving with lactic acid (85%). Plates were incubated under aerobic conditions at 32°C for 48 h. Lactic acid bacteria (d 0, 3, and 8 only) were enumerated on De Man, Rogosa, Sharpe agar (Oxoid CM0361) and incubated at 32°C for 48 h. Dried forage and silage samples were ground through a 1-mm screen of a Cyclone Sample Mill (Udy Corp., Fort Collins, CO). Samples were analyzed for N by total combustion of the sample (Leco CNS 2000 Analyzer, Leco, St. Joseph, MI). Aerobic stability was determined on silages that had been ensiled for 256 d by exposing 3 kg of loose silage to air in 20-L buckets. The buckets were covered with cheesecloth to allow air to infiltrate the silage mass and prevent the silage from drying out and becoming contaminated. A thermocouple wire was inserted in the middle of the silage mass. The wire was attached to a data logger (model number CR10X, Campbell Scientific Inc., Logan, UT) that recorded the temperature every 10 min. Data were averaged every 2 h and aerobic stability was defined as the number of hours the silage remained stable before a 2°C increase in temperature above the ambient temperature (
      • Kung Jr, L.
      • Ranjit N.K.
      The effect of Lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage.
      ).

      Lactation Trial

      This study was approved by the Animal Care and Use Committee of the College of Agriculture and Natural Resources, University of Delaware, and followed the care and management of animals as per accepted guidelines (
      Agricultural Animal Care and Use Committee
      Handbook for Agricultural Animal Care and Use in Research and Teaching.
      ). Thirty lactating cows (4 primiparous and 26 multiparous), averaging 118 ± 70 DIM and 38 ± 16 kg of milk/d, were housed in a free-stall barn and fed via Calan gates (American Calan, Northwood, NH) to measure individual daily intake. Cows were fed a TMR once daily that consisted of (on a DM basis) 25% corn silage, 15% alfalfa silage, 10% alfalfa hay, and 50% concentrate (Tables 1 and 2). The rations were balanced to meet
      NRC.
      Nutrient Requirements of Dairy Cattle.
      requirements for cows producing 40 kg of milk/d, 3.5% fat, and 3.1% protein and weighing 680 kg. Cows had access to fresh water at all times. Cows were allowed to exercise twice daily in a dirt lot for approximately 1 to 2 h. All cows were supplemented with a dry additive mixture of essential plant oils blended in limestone. Fifty grams of the limestone-essential oil blend (Crina) was mixed by hand into the TMR of all cows to provide a daily intake of 0.6 g of essential oils/cow per d during the 14-d pretreatment period. This was done to accustom all cows to the smell and taste of the essential oils. At the end of the pretreatment period, cows were blocked by lactation number, pretreatment milk production, DIM, and lactation number and then randomly allocated to 1 of 2 treatments. During a 9-wk treatment period, one-half of the cows received the base TMR without supplementation, and the remaining half of the cows were fed the same TMR supplemented with essential oils to provide an intake of 1.2 g/cow per d. Control cows were supplemented with only the lime- stone carrier.
      Table 1Composition (%, DM basis) of concentrate mixed into the TMR during the lactation experiment
      IngredientAmount, %
      Corn meal (fine)42.42
      Distillers dried grains10.72
      Corn gluten feed10.72
      Soybean roasted7.18
      Soybean meal (47.5%)5.58
      Wheat middlings5.48
      Molasses blend
      Contained (DM basis) 0.5% Ca, 0.03g of Cu/kg, 5.0% crude fat, 3.0% CP, 0.50g of I/kg, 0.40% Mg, 1.50 mcal/kg of NEL, 45.5% NSC, 3.2% K, 0.70% S, 63% total digestible nutrients, 0.50% P, 0.01g of Zn/kg, 0.94 undegradable intake protein.
      4.16
      Corn starch2.69
      Protein blend
      Dried blood meal, hydrolyzed poultry feathers, fishmeal, calcium carbonate, and ethoxyquin.
      1.84
      Fat blend
      Feed-grade combination of tallow, poultry, and porcine fats.
      1.67
      Sesquicarbonate2.04
      Limestone (37%)1.76
      Corn gluten (60% CP)1.50
      Salt0.96
      Magnesium oxide0.57
      Urea0.51
      Selenium (0.06%)0.10
      Trace minerals
      Contained 2.1% Ca, 1.6g of Co/kg, 40g of Cu/kg, 4.0g of I/kg, 6.0g of Fe/kg, 150g of Mn/kg, 13.0% S, 160g of Zn/kg.
      0.05
      Vitamins
      Contained (DM basis) 26,400 kIU of vitamin A/kg, 8,800 kIU/kg of vitamin D, and 165,000 IU of vitamin E/kg.
      0.05
      1 Contained (DM basis) 0.5% Ca, 0.03 g of Cu/kg, 5.0% crude fat, 3.0% CP, 0.50 g of I/kg, 0.40% Mg, 1.50 mcal/kg of NEL, 45.5% NSC, 3.2% K, 0.70% S, 63% total digestible nutrients, 0.50% P, 0.01 g of Zn/kg, 0.94 undegradable intake protein.
      2 Dried blood meal, hydrolyzed poultry feathers, fishmeal, calcium carbonate, and ethoxyquin.
      3 Feed-grade combination of tallow, poultry, and porcine fats.
      4 Contained 2.1% Ca, 1.6 g of Co/kg, 40 g of Cu/kg, 4.0 g of I/kg, 6.0 g of Fe/kg, 150 g of Mn/kg, 13.0% S, 160 g of Zn/kg.
      5 Contained (DM basis) 26,400 kIU of vitamin A/kg, 8,800 kIU/kg of vitamin D, and 165,000 IU of vitamin E/kg.
      Table 2Measured nutrient composition of the individual feeds (average value of 3 composite samples) and TMR (average value from 9 weekly composite samples) fed to lactating cows throughout the lactation study
      ItemAlfalfa hayAlfalfa silageCorn silageConcentrateControl TMRBEO
      TMR supplemented with a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) to supply 1.2 g/cow per d.
      TMR
      DM, %88.051.835.688.263.8 ± 2.5964.0 ± 1.62
      CP, %16.2419.818.9620.2916.36 ± 0.5816.43 ± 0.78
      NEL, mcal/kg1.141.341.561.981.52 ± 0.041.50 ± 0.04
      ADF, %45.6237.4629.256.4425.84 ± 2.2525.97 ± 2.85
      NDF, %52.6443.4546.7117.6735.44 ± 2.0935.32 ± 2.37
      Ash, %7.0112.043.865.678.14 ± 0.488.32 ± 1.56
      Starch, %38.6718.79 ± 1.8718.88 ± 1.79
      NFC, %21.2419.7737.6544.5637.04 ± 1.6636.93 ± 1.82
      Ca, %0.821.680.200.900.92 ± 0.011.12 ± 0.44
      P, %0.290.330.210.550.35 ± 0.010.35 ± 0.02
      Mg, %0.180.380.160.560.30 ± 0.020.31 ± 0.02
      K, %2.383.021.010.981.46 ± 0.061.51 ± 0.14
      Particle size distribution,
      As determined on a Penn State Particle Size Separator (Pennsylvania State University, State College, PA) with 4 screens.
      %
       Screen 111.62 ± 2.5411.32 ± 2.92
       Screen 223.78 ± 3.3722.60 ± 2.70
       Screen 341.38 ± 3.8942.10 ± 4.71
       Bottom pan21.11 ± 2.5622.34 ± 3.95
      1 TMR supplemented with a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) to supply 1.2 g/cow per d.
      2 As determined on a Penn State Particle Size Separator (Pennsylvania State University, State College, PA) with 4 screens.
      A computer recorded milk production from each cow twice daily at approximately 0600 and 1800 h. Once weekly, milk was sampled proportionately to milk yield from consecutive p.m. and a.m. milkings and analyzed for fat, protein, lactose, MUN, and SCC (Dairy One, University Park, PA). Body weights were recorded on 2 consecutive days at the beginning and end of the study. Body condition scores (
      • Wildman E.E.
      • Jones G.M.
      • Wagner P.E.
      • Boman R.L.
      • Troutt Jr H.F.
      • Lesch T.N.
      A dairy cow body condition scoring system and its relationship to selected production characteristics.
      ) were recorded at the beginning and end of the study. Feed offered and refused was measured daily. Samples of all feeds were collected 3 times weekly and composited on a weekly basis. Once weekly, a representative sample of each TMR was subjected to quantitative measurements of particle size by using a Penn State Particle Size Separator (Pennsylvania State University, State College, PA). Dry matter of the feed samples was determined in a forced-air oven at 60°C for 48 h. Dietary ingredients were adjusted on a weekly based on the DM content of the feeds. After drying, feed samples were ground through a Wiley mill (1-mm screen, Arthur H. Thomas, Philadelphia, PA) and analyzed for laboratory DM (100°C oven for 24 h), NDF by using sulfite and amylase (
      • Van Soest P.J.
      • Robertson J.B.
      • Lewis B.A.
      Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
      ), and ADF (
      • Robertson J.B.
      • Van Soest P.J.
      The detergent system of analysis and it application to human foods.
      ). Total N was determined as described previously for corn silages. Starch was analyzed by the Cumberland Valley Analytical Services laboratory (Maugansville, MD) and the mineral content of the TMR was analyzed by inductively coupled plasma emission spectrometry on a composite sample from each period (University of Delaware Soil Testing Program, Newark, DE).

      In Vitro Ruminal Fermentation

      The effect of the blend of essential oils on ruminal in vitro fermentation was tested by using the system described by
      • Goering H.K.
      • Van Soest P.J.
      Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications).
      with some modifications. Rumen inoculum was withdrawn from a fistulated Holstein steer, fed once daily for ad libitum intake of a TMR composed of 35% corn silage, 15% alfalfa silage, 5% alfalfa hay, and approximately 45% of a concentrate mix (DM basis). Ruminal fluid was strained through 4 layers of cheesecloth, mixed in a 1-to-4 proportion with a phosphate-bicarbonate buffer, and purged constantly with anaerobic-grade CO2. The primary modification included using 90-mL polycarbonate tubes containing 50 mL of culture fluid with 0.5 g of the same diet used during the lactation experiment and ground through a 2-mm screen. Five replicate tubes were prepared for each treatment. The tubes were purged with CO2 before sealing with butyl rubber stoppers fitted with gas-release valves. Tubes were placed in a heated (39°C) orbital shaker (100 rpm). Treatments were nothing, the dry blend of essential oils (Crina) at a physiological dose (0.044 mg/kg of feed DM), and the blend of essential oils at a level 25 times greater (1.111 mg/kg of feed DM). Based on the experimental conditions described previously, feeding the physiological dose would provide a cow consuming 27 kg of DM/d with 1.2 g of active ingredient/d. The blend of essential oils was dissolved in 50% ethanol, and 1 mL was added to each tube. The equivalent amount of ethanol alone was added to control tubes. Samples were taken at 12 h. A 10-mL aliquot was sampled from each tube, and ruminal fluid pH was measured immediately. Ruminal fluid was then acidified with 50 μL of 50% H2SO4 and frozen (−20°C) before analysis for VFA. Volatile fatty acids were determined by GC as described by
      • Kung Jr, L.
      • Ranjit N.K.
      The effect of Lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage.
      .

      Statistical Analysis

      Data from the silage experiment, the lactation trial, and the ruminal in vitro fermentation were analyzed as completely randomized designs by using the general linear models procedure (SAS Institute Inc., Cary, NC). Data from the pretreatment period of the lactation study were used for covariate adjustment. Tukey's test (
      • Snedecor G.W.
      • Cochran W.G.
      Statistical Methods.
      ) was used to test treatment means (P < 0.05) of the silage experiment and lactation trial. For the rumen in vitro data, contrasts were constructed to compare 1) the control versus the addition of essential oils, and 2) the addition of essential oils at a physiological dose versus at a level 25 times greater. Significance was defined at P < 0.05.

      Results and Discussion

      Silage Experiment

      The chemical and microbial composition of freshly chopped forages is shown in Table 3. The chemical and microbial compositions of silages after 3 and 8 d are not shown because the addition of the essential oils had only minor effects on the measured variables compared with untreated silage. We hypothesized that the blend of essential oils used in the current study might be useful as a silage additive. Specifically, past research has shown that compounds such as limonene (
      • Figueiredo A.R.
      • Campos F.
      • de Freitas V.
      • Hogg T.
      • Couto J.A.
      Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii.
      ) and eugenol (
      • Kim H.G.
      • Cho J.H.
      • Jeong E.Y.
      • Lim J.H.
      • Lee S.H.
      • Lee H.S.
      Growth-inhibiting activity of active component isolated from Terminalia chebula fruits against intestinal bacteria.
      ) had no effects on lactic acid bacteria and thus would not pose a problem for these beneficial bacteria in silages. However, thymol has been shown to inhibit the growth of D. hansenii (
      • Curtis O.F.
      • Hetty K.S.
      • Cassagnol G.
      • Peleg M.
      Comparison of the inhibitory and lethal effects of synthetic versions of plant metabolites (anethole, carvacrol, eugenol, and thymol) on food spoilage yeast (Debaromyces hansenii).
      ) and species of Pichia and Candida (
      • Falcone P.
      • Speranza B.
      • Nobile M.A.
      • Corbo M.R.
      • Sinigaglia M.
      A study on the antimicrobial activity of thymol intended as a natural preservative.
      ). These yeasts have been identified as initiators of aerobic spoilage in silages (
      • Pahlow G.
      • Muck R.E.
      • Driehuis F.
      • Oude Elferink S.J.W.H.
      • Spoelstra S.F.
      Microbiology of ensiling.
      ). Similarly, limonene has been shown to be anti-fungal against species of Candida and Aspergillus flavus (
      • Adegoke G.O.
      • Iwahashi H.
      • Komatsu Y.
      • Obuchi K.
      • Iwahashi Y.
      Inhibition of food spoilage yeasts and aflatoxigenic moulds by monoterpenes of the spice Aframomum danielli.
      ). In the current study, the blend of essential oils did not affect the numbers of yeasts and molds, enterobacteria, or lactic acid bacteria compared with untreated silage during the early stages of ensiling (3 and 8 d, data not shown) and these findings were still found after 256 d of ensiling (Table 4). The exact reason for the lack of a measurable antifungal effect is unknown because the concentration of essential oils was within the range at which inhibition has been shown in pure culture, but this may have been due to inactivation of the essential oils in the complex environment of silage. When compared with untreated silage, the addition of the low and high doses of the essential oil mixture had no effect on DM recovery, fermentation end products, nutrient content, yeasts and molds, or aerobic stability. Addition of the buffered propionic acid additive moderately depressed the production of lactic and acetic acids when compared with untreated silage after 256 d. As expected, concentrations of propionic acid were greatest for this treatment, which resulted in the smallest number of yeasts among treatments. However, because the depression in yeasts was relatively small, there was no effect of the additive on the aerobic stability of corn silage. Buffered propionic acid additives have decreased the number of yeasts and improved aerobic stability in past studies (
      • Kung Jr, L.
      • Sheperd A.C.
      • Smagala A.M.
      • Endres K.M.
      • Bessett C.A.
      • Ranjit N.K.
      • Glancey J.L.
      The effect of propionic acid-based preservatives on the fermentation and aerobic stability of corn silage and a total mixed ration.
      ;
      • Kung Jr, L.
      • Ranjit N.K.
      The effect of Lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage.
      ), and the reason for a lack of a response in the current study is unknown.
      Table 3The pH, chemical (DM basis) composition, and microbial composition (wet basis) of freshly chopped whole-plant corn after treatment but before ensiling (analysis from a single sample for each treatment)
      TreatmentControl
      Untreated forage.
      BEO,
      Forage treated to achieve 40mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) per kilogram of fresh forage.
      BEO,
      Forage treated to achieve 80mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd.) per kilogram of fresh forage.
      Buffered propionic acid
      A buffered propionic acid-based additive (Ultra Chem, Kemin Industries, West Des Moines, IA, added at 0.2% of fresh weight).
      DM, %28.4629.9128.5933.69
      pH5.365.395.405.04
      Water-soluble carbohydrates, %6.537.016.846.46
      Ammonia-N, %0.0820.0780.0860.087
      Yeasts, log10 cfu/g6.977.246.916.20
      Molds, log10 cfu/g6.296.116.096.00
      Enterobacteria, log10 cfu/g7.677.597.697.62
      Lactic acid bacteria, log10 cfu/g7.437.437.347.40
      1 Untreated forage.
      2 Forage treated to achieve 40 mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) per kilogram of fresh forage.
      3 Forage treated to achieve 80 mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd.) per kilogram of fresh forage.
      4 A buffered propionic acid-based additive (Ultra Chem, Kemin Industries, West Des Moines, IA, added at 0.2% of fresh weight).
      Table 4Dry matter recovery, silage pH, chemical (DM basis) composition, microbial composition (wet basis), and aerobic stability of corn silages after 256 d of ensiling
      TreatmentControl
      Untreated forage.
      BEO,
      Forage treated to achieve 40mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) per kilogram of fresh forage.
      BEO,
      Forage treated to achieve 80mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd.) per kilogram of fresh forage.
      Buffered propionic acid
      A buffered propionic acid-based additive (Ultra Chem, Kemin Industries, West Des Moines, IA, added at 0.2% of fresh weight).
      SEM
      DM recovery, %89.986.488.688.11.1
      DM, %26.1b26.4b25.8b30.4
      Means within rows with unlike superscripts differ (P<0.05).
      0.3
      pH3.72
      Means within rows with unlike superscripts differ (P<0.05).
      3.71
      Means within rows with unlike superscripts differ (P<0.05).
      3.66
      Means within rows with unlike superscripts differ (P<0.05).
      b
      3.64b0.02
      Lactic acid, %5.50
      Means within rows with unlike superscripts differ (P<0.05).
      5.34
      Means within rows with unlike superscripts differ (P<0.05).
      5.70
      Means within rows with unlike superscripts differ (P<0.05).
      4.53b0.26
      Acetic acid, %2.20
      Means within rows with unlike superscripts differ (P<0.05).
      1.98
      Means within rows with unlike superscripts differ (P<0.05).
      b
      2.14
      Means within rows with unlike superscripts differ (P<0.05).
      1.86b0.07
      Propionic acid, %<0.01b<0.01b<0.01b0.53
      Means within rows with unlike superscripts differ (P<0.05).
      <0.01
      Ammonia-N, %0.229
      Means within rows with unlike superscripts differ (P<0.05).
      b
      0.226
      Means within rows with unlike superscripts differ (P<0.05).
      b
      0.248
      Means within rows with unlike superscripts differ (P<0.05).
      0.201b0.015
      Water soluble carbohydrates, %2.34b2.67b2.19b3.29
      Means within rows with unlike superscripts differ (P<0.05).
      0.15
      Yeasts, log10 cfu/g5.16
      Means within rows with unlike superscripts differ (P<0.05).
      5.41
      Means within rows with unlike superscripts differ (P<0.05).
      5.18
      Means within rows with unlike superscripts differ (P<0.05).
      4.45b0.22
      Molds, log10 cfu/g4.474.574.513.620.31
      Aerobic stability,
      The number of hours silage remained stable after exposure to air before a 2°C increase in temperature above ambient temperature.
      h
      47.545.349.559.54.2
      a Means within rows with unlike superscripts differ (P < 0.05).
      1 Untreated forage.
      2 Forage treated to achieve 40 mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) per kilogram of fresh forage.
      3 Forage treated to achieve 80 mg of a blend of essential oils (Crina, DSM Nutritional Products Ltd.) per kilogram of fresh forage.
      4 A buffered propionic acid-based additive (Ultra Chem, Kemin Industries, West Des Moines, IA, added at 0.2% of fresh weight).
      5 The number of hours silage remained stable after exposure to air before a 2°C increase in temperature above ambient temperature.

      Lactation Trial

      The average composition of the TMR fed to cows throughout the study is shown in Table 2. The diets were similar in nutrient composition. The distribution of feed particles suggested that the diets were marginal in effective fiber because the percentage of particles on the second screen was approximately 23%, whereas recommendations suggest a 30 to 50% distribution on this screen (
      • Heinrichs J.A.
      • Kononoff P.
      Evaluating particle size of forages and TMR using the new Penn State forage separator. DAS-020-42.
      ).
      Data from the lactation experiment are shown in Table 5. Dry matter intake was greater from cows fed essential oils (28.3 kg/d) compared with cows fed the control diet (26.4 kg/d). Similarly, DMI expressed as a percentage of BW was also greater for cows fed the essential oils. These findings are in contrast to a depression in DMI when cows were fed Crina, as reported by
      • Tassoul M.D.
      • Shaver R.D.
      Efficacy of essential oils as dietary supplements for dairy cows.
      , but cows were in a much earlier stage of lactation in their study. In our study, cows fed a TMR supplemented with essential oils tended (P < 0.16) to produce more milk (41.7 kg/d) than cows fed the control diet (39.8 kg/d). The percentage of fat in milk was numerically greater, although not statistically, in cows supplemented with essential oils. This finding led to an increase in milk fat production for cows fed the supplement (1.24 vs. 1.13 kg/d). Furthermore, 3.5% FCM was greater in cows fed essential oils (38.2 vs. 35.5 kg/d). Several studies have also evaluated the effectiveness of feeding the same blend of essential oils (Crina) as was used in the current study.
      • Offer N.W.
      • Bell J.F.
      • Roberts D.J.
      The effect of feeding an essential oil feed additive on dairy cattle performance.
      fed cows 3 doses of Crina (0.5, 1.0, and 2.0 g/d) and reported an increase of 1.2, 1.6, and 1.8 kg of FCM/d, respectively, for the 3 doses. In a field study,
      • Varga G.
      • Block E.
      • Williams P.
      • Cassidy T.W.
      • Losa R.
      Effect of Crina Ruminants, a mixture of essential oil components, on continuous culture fermentation and milk production of lactating cows.
      reported that feeding Crina improved FCM production by 1.6 kg/d. In studies by
      • Benchaar C.
      • Petit H.V.
      • Berthiaume R.
      • Ouellet D.R.
      • Chiquette J.
      • Chouinard P.Y.
      Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production, and milk composition in dairy cows.
      ,
      • Benchaar C.
      • Petit H.V.
      • Berthiaume R.
      • Ouellet D.R.
      • Chiquette J.
      • Chouinard P.Y.
      Effects of essential oils on digestion, ruminal fermentation, rumen microbial populations, milk production, and milk composition in dairy cows fed alfalfa silage or corn silage.
      ), feeding the same product we used depressed FCM, but the number of cows per treatment was limited in those studies. Recently,
      • Tassoul M.D.
      • Shaver R.D.
      Efficacy of essential oils as dietary supplements for dairy cows.
      reported that feeding Crina in early lactation did not affect milk production.
      Table 5Least squares means of performance data from lactating cows fed a TMR with or without a blend of essential oils
      ItemControlBEO
      TMR supplemented with a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) to supply 1.2 g/cow per d.
      SEM
      DMI, kg/d26.4b28.3
      Means in rows with unlike superscripts differ P<0.05
      0.5
      Milk
       Yield, kg/d39.8
      Means in rows with unlike superscripts differ P<0.16
      41.7d0.9
       Fat, %2.892.990.09
       Fat, kg/d1.13b1.24
      Means in rows with unlike superscripts differ P<0.05
      0.03
       Protein, %3.113.070.04
       Protein, kg/d1.231.270.02
       Urea N, mg/dL11.111.60.4
      Somatic cells/mL, × 1,00024224340
      3.5% FCM, kg/d35.5
      Means in rows with unlike superscripts differ P<0.05
      38.2b0.8
      DMI, % of BW3.98b4.23
      Means in rows with unlike superscripts differ P<0.05
      0.08
      Feed efficiency, DMI:FCM1.361.350.03
      BCS3.23.20.1
      Change in BCS
      Change over the 9-wk treatment period.
      0.070.160.06
      BW, kg6626718
      Change in BW, kg/d0.530.860.28
      a Means in rows with unlike superscripts differ P < 0.05
      c Means in rows with unlike superscripts differ P < 0.16
      1 TMR supplemented with a blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) to supply 1.2 g/cow per d.
      2 Change over the 9-wk treatment period.
      Overall, the milk fat content of the current study was low (approximately 2.9%) and may have been a reflection of the low percentage of particles on the second screen of the particle separator and the fat supplement, which contained tallow, poultry, and porcine fats. There were, however, no observed signs of laminitis during the study. Milk protein percentage, milk protein, MUN, and SCC were not affected by treatment. Feed efficiency was similar between treatments. Body score, change in body score, BW, and change in BW were also similar between treatments.

      In Vitro Fermentation

      After 12 h, the addition of essential oils to in vitro ruminal fermentations had no effect on culture pH and total concentration of VFA (Table 6). Addition of essential oils decreased the molar proportion of acetic, butyric, and valeric acids, but increased the molar proportion of propionic acid. When compared with the moderate dose of essential oils, the larger dose increased the molar proportions of acetic, isobutyric, butyric, isovaleric, and valeric acids but decreased the molar proportion of propionic acid. Effects of essential oils on ruminal fermentation have been extremely variable (
      • Calsamiglia S.
      • Busquet M.
      • Cardozo P.W.
      • Castillejos L.
      • Ferret A.
      Essential oils as modifiers of rumen microbial fermentation.
      ) and may be affected by diet and rumen pH (
      • Castillejos L.
      • Calsamiglia S.
      • Ferret A.
      • Losa R.
      Effects of a specific blend of essential oil compounds and the type of diet on rumen microbial fermentation and nutrient flow from a continuous culture system.
      ), the system in which they are evaluated (in vivo, batch in vitro, or continuous culture;
      • Benchaar C.
      • Petit H.V.
      • Berthiaume R.
      • Ouellet D.R.
      • Chiquette J.
      • Chouinard P.Y.
      Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production, and milk composition in dairy cows.
      ), and the length of the fermentation process (
      • Cardozo P.W.
      • Calsamiglia S.
      • Ferret A.
      • Kamel C.
      Screening for the effects of a natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle.
      ).
      Table 6Effects of a blend of essential oils (BEO) on ruminal fermentation after 12 h of incubation
      Contrast, P-value
      ItemControlBEO,
      A blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) at a physiological dose of 0.044mg/kg of feed (DM basis).
      BEO,
      A blend of essential oils (Crina, DSM Nutritional Products Ltd.) at a dose of 1.111mg/kg of feed (DM basis).
      25×
      SEMControl vs. BEO, 1× and BEO, 25×BEO, 1× vs. BEO, 25×
      pH6.786.836.830.07<0.25<0.87
      VFA, molar %
       Acetic acid67.3364.5566.411.23<0.02<0.03
       Propionic acid22.9727.0824.071.75<0.02<0.02
       Isobutyric acid1.521.331.610.20<0.61<0.04
       Butyric acid5.524.955.290.22<0.01<0.03
       Isovaleric acid1.390.991.390.24<0.17<0.03
       Valeric acid1.271.101.230.07<0.02<0.02
      Total VFA, mM76.4572.1775.745.12<0.39<0.29
      1 A blend of essential oils (Crina, DSM Nutritional Products Ltd., Parsippany, NJ) at a physiological dose of 0.044 mg/kg of feed (DM basis).
      2 A blend of essential oils (Crina, DSM Nutritional Products Ltd.) at a dose of 1.111 mg/kg of feed (DM basis).

      Conclusions

      A blend of essential oils added to corn forage at ensiling did not affect the microorganisms, the fermentation process, or the aerobic stability of corn silage. When added as a physiological dose to in vitro ruminal fermentations, the blend of essential oils reduced the molar proportion of acetate but increased the molar proportion of propionate, which may partially explain why, when fed to lactating dairy cows, the blend of essential oils increased DMI and 3.5% FCM production.

      Acknowledgments

      The authors thank the farm staff of the University of Delaware Dairy for harvesting the crops and Richard Morris, dairy herdsman, for care of the animals.

      Supplementary data

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