In vitro and in vivo potential of a blend of essential oil compounds to improve rumen fermentation and performance of dairy cows

Animal Feed Science and Technology 251 (2019) 176–186

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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

In vitro and in vivo potential of a blend of essential oil compounds to improve rumen fermentation and performance of dairy cows

T

⁎

M. Jocha,b, , V. Kudrnaa, J. Haklc, M. Božikd, P. Homolkaa,b, J. Illeka, Y. Tyrolováa, A. Výbornáa a

Department of Nutrition and Feeding of Farm Animals, Institute of Animal Science, Přátelství 815, 104 00 Prague, Czech Republic Department of Microbiology, Nutrition and Dietetics, Czech University of Life Sciences, Kamýcká 129, 165 00 Prague, Czech Republic c Department of Forage Crops and Grassland Management, Czech University of Life Sciences, Kamýcká 129, 165 00 Prague, Czech Republic d Department of Quality of Agricultural Products, Czech University of Life Sciences, Kamýcká 129, 165 00 Prague, Czech Republic b

A R T IC LE I N F O

ABS TRA CT

Keywords: Feed additive Essential oil Thymol Dairy cow Rumen fermentation Methane

The objective of this study was to evaluate in vitro and in vivo potential of a specific commercial blend of essential oil active compounds (BEO) to improve rumen fermentation and general dairy cow performance. First, the main active components of the BEO were identified and quantified. Then, the in vitro experiment was conducted using 24 h batch incubation of buffered rumen fluid. The BEO was added at 0, 20, 100, 200, 600, and 1000 mg/l of culture fluid, with substrate containing forage and concentrate at a ratio of 60:40 dry matter (DM) basis. The concentrations of 600 and 1000 mg/l decreased (P < 0.05) methane production per dry matter incubated (DMi) by 5.7% and 17.1%, respectively, and a concentration of 1000 mg/l decreased ammonia-N concentration by 10.0%. However, these reductions were accompanied by a decrease (P < 0.05) in apparent dry matter disappearance (aDMd), and lowered (P < 0.05) the net production of volatile fatty acids (nVFA) indicating, that there were no beneficial selective inhibitory properties of BEO supplementation. For the in vivo experiment, 30 lactating Holstein cows (two primiparous and 28 multiparous) in mid-lactation were randomly assigned to two treatments. During a 15 week period, cows in the control group (CON; n = 15) were fed the basal diet with no additive, and cows in the other group (BEO; n = 15) received the same diet supplemented with 1.2 g/cow/ d BEO. Addition of BEO did not affect (P = 0.218) DM intake and milk yield (P = 0.102). For feed efficiency (P = 0.047) and body weight (P = 0.014), the treatment × time interactions were observed, with cows fed BEO having a lower average feed efficiency and a higher average body weight. BEO treatment tended to decrease the proportion of milk fat (P = 0.072), without affecting other milk quality parameters. BEO supplemented cows had lower VFA concentrations in rumen fluid (P = 0.006), and correspondingly, pH values were higher (P = 0.018). No differences were detected between the treatments for proportions of individual VFA and ammonia-N concentration. The responses of dairy cows on BEO were more pronounced after the 5th week of supplementation. This indicates the necessity of a longer adaptation period than commonly used in studies evaluating the effects of essential oils (EO). Due to energetic shift toward body weight

Abbreviations: ADFom, acid detergent fiber expressed exclusive of residual; aDMd, apparent dry matter disappearance; aNDFom, neutral detergent fiber assayed with a heat stable amylase and expressed exclusive of residual ash; BEO, blend of essential oil active compounds; CCM, corn cob mix silage; CON, control group of animals not supplemented with BEO; DIM, days in milk; DM, dry matter; DMi, dry matter incubated; EO, essential oil; MUN, milk urea nitrogen; NFC, non-fiber carbohydrates; nVFA, net production of volatile fatty acids; TGP, total gas production; TMR, total mixed ratio; VFA, volatile fatty acids ⁎ Corresponding author at: Department of Nutrition and Feeding of Farm Animals, Institute of Animal Science, Přátelství 815, 104 00 Prague, Czech Republic. E-mail address: [email protected] (M. Joch). https://doi.org/10.1016/j.anifeedsci.2019.03.009 Received 4 September 2018; Received in revised form 8 February 2019; Accepted 24 March 2019 0377-8401/ © 2019 Elsevier B.V. All rights reserved.

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gain additional attention should be paid to the systemic effects of EO. Generally, results from this study suggest that mid-lactation dairy cows did not benefit from being supplemented with BEO.

1. Introduction Antibiotic ionophores were successful in reducing energy and protein losses in the rumen (Nagaraja et al., 1997). However, due to appearance of residues and resistant strains of bacteria, the use of antibiotics as feed additives resulted in reduced social acceptance, which led to the ban on their use in the European Union since January 2006 (Calsamiglia et al., 2007). Consequently, the demand for more natural and safe alternative feed additives that can be used to improve animal production rapidly increased (Jouany and Morgavi, 2007). In line with this, in recent years, numerous studies have been conducted to evaluate essential oils (EO) and their active compounds as natural feed additives in ruminant nutrition. EO are volatile aromatic compounds produced by plants (herbs and spices) as complex mixtures of secondary metabolites, and possess, among others, antiseptic and antimicrobial activities (Cobellis et al., 2016a). There is some evidence of positive effects of EO on rumen fermentation. For example, EO may result in decrease intra-ruminal nitrogen turnover and nitrogen excretion (Patra and Yu, 2014), EO may slow down degradation of starch-rich substrates (Hart et al., 2008), and may inhibit methanogenesis (Patra and Saxena, 2010). Most of the described effects are based on the influence of EO on the microbial population in the rumen and the rumen fermentation (Flachowsky and Lebzien, 2012). However, the positive effects of EO seem to be inconsistent, and are often accompanied by negative effects on fiber degradation and VFA production. These adverse effects could be attributed to the broad and often non-specific antimicrobial activities of EO within the rumen (Cobellis et al., 2016a). Despite these facts, there are commercially available feed additives based on EO active compounds with declared positive effects on rumen fermentation and digestion. Several in vitro or in vivo studies (Benchaar and Greathead, 2011; Cobellis et al., 2016a) and a few studies that test for effects in vivo and in vitro simultaneously (Kung et al., 2008; Castro-Montoya et al., 2015) have examined the potential of these products to improve rumen fermentation efficiency, with variable results, even at same doses of products. Differences in results could be attributable to (among other things) differences in contents of key substances, as EO active compounds have a low chemical stability and high volatility (Cobellis et al., 2016a). Unfortunately, in the studies using commercial blends of essential oil active compounds (BEO), active components in administrated products are not usually precisely characterized and quantified, in spite of the different effects of particular compounds (Benchaar and Greathead, 2011). Moreover, regarding EO, in vitro studies outnumbered in vivo studies in general, but regarding commercial available BEO, dose-response in vitro studies are scarce (Cobellis et al., 2016a). Direct comparison of results from in vitro and in vivo experiments may help better distinguish between antimicrobial and systemic (e.g. metabolic) effects of EO since only effects on rumen microbial populations are estimated in in vitro experiment. In addition, direct comparison may also help understanding how results from in vitro experiments could be used in the prediction of EO effects in animals. Therefore, the objectives of this study were: 1) to identify and quantify the main active components of a commercial blend of essential oil compounds (BEO), 2) to identify the minimal effective concentration and to evaluate dose-response effects of BEO on fermentation parameters using in vitro 24 h batch incubation of buffered rumen fluid, and 3) to investigate the in vivo effects of dietary addition of BEO on feed intake, feed efficiency, milk production and rumen fermentation of dairy cows in mid-lactation. 2. Materials and methods The experiments were designed and performed according to European and Czech laws. The protocol was approved by the Institutional Animal Care and Use Committee of the Institute of Animal Science in Prague. 2.1. Analyses of commercial blend of essential oil active compounds (BEO) The BEO consisted of an active ingredient, an aromatizing mixture, and an absorbing support. Declared ranges of EO active compounds were as follows: cresols 50–100 mg/g, thymol 50–105 mg/g, limonene 40–105 mg/g, vanillin 20–60 mg/g, guaiacol 20–45 mg/g, eugenol 10–30 mg/g and salicylates 10–30 mg/g. In total, the declared content of volatile fraction was 250–400 mg/g of product (Rossi, 1995). One hundred milligrams of BEO was extracted using 10 ml of hexane with an internal standard (octadecane, concentration 0.1 mg/ml) on an orbital shaker for 10 min. Then, 1 ml of extract was filtered through a membrane filter into a chromatographic vial, and immediately measured by gas chromatography with flame ionization detector (GC-FID) Agilent 7890A (Agilent Technologies, Palo Alto, CA, USA) using an HP-5MS column (30 m ×0.25 mm, 0.25 μm film thickness). One microliter of the sample was injected in split mode 1:12, at an injector temperature of 250 °C, with the oven program starting at 60 °C. The temperature was increased at a rate of 3 °C/min to a maximum of 231 °C, where it was kept constant for 10 min. The carrier gas used was nitrogen (constant flow 1 ml/min, 99.999% purity), and the detector temperature was 250 °C. Compounds were identified by gas chromatography followed by mass spectrometry (GC–MS). Analyses were carried out using an Agilent 7890A GC coupled to an Agilent MSD5975C MS detector (Agilent Technologies, Palo Alto, CA, USA) with a HP-5MS column (30 m ×0.25 mm, 0.25 μm film thickness), and an electron ionization energy of 70 eV. Analysis was measured in SCAN mode, and the mass range was 40–400 m/z. The identification of constituents was based on a comparison of their mass spectra and relative retention indices (RI) against the National Institute of 177

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Standards and Technology Library (NIST, USA), as well as authentic analytical standards (Adams, 2007). The same method as for GCFID was employed. 2.2. In vitro experiment 2.2.1. Experimental design and treatments To identify dose-response effects and threshold concentration for manifestation of effects on parameters of 24 h in vitro batch incubation of rumen fluid, six concentrations (0, 20, 100, 200, 600, and 1000 mg/l of culture fluid) of BEO were evaluated. The BEO was dissolved in ethanol, and an individual stock solution was prepared for each concentration. The stock solutions were homogenized in tubes sealed with parafilm caps using a TissueRuptor (Qiagen, Crawley, United Kingdom) due to the presence of insoluble components in the BEO. The concentrations of BEO were tested in triplicate within the incubation, and incubations were repeated on three separate days (runs). Three blanks (no substrate, no additive) were always included with each run. 2.2.2. Substrate and rumen fluid The experimental substrate consisted (DM basis) of corn silage (300 g/kg), alfalfa silage (300 g/kg), and concentrate mixture (400 g/kg). The chemical composition per kg DM of the substrate was 916 g organic matter, 162 g crude protein, 45 g ether extract, 233 g starch, 331 g aNDFom, and 215 g ADFom. Fresh rumen content was manually collected from two rumen-fistulated lactating Holstein cows (averaging 6 ± 1 parity; 138 ± 10 days in milk, DIM; 828 ± 86 kg body weight, and 34 ± 2 kg of milk/d) before morning feeding. The cows were fed a total mixed ratio (TMR) ad libitum (offered fresh two times per day) composed of (DM basis) corn silage (210 g/kg), corn cob mix silage (CCM; 205 g/kg), alfalfa silage (200 g/kg), brewers grain (97 g/kg), and a concentrate mixture (288 g/kg). Rumen content from each cow was placed into a pre-warmed 500 ml CO2-flushed insulated flask, leaving no headspace, and transported to the laboratory. Rumen fluid was obtained by straining rumen content through a sieve with a pore size of 315 μm under continuous CO2 flushing. Equal volumes of the rumen fluid collected from each of the cows were combined for inoculum preparation. Time from collection to inoculation did not exceed 45 min. 2.2.3. In vitro incubation The in vitro batch incubations were conducted as described by Fievez et al. (2005). Briefly, 250 mg of the dried substrate was incubated in a 120 ml capacity gastight serum bottles with 25 ml of buffered rumen fluid. Before inoculation, the rumen fluid was mixed at a 1:4 ratio with a phosphate buffer (per liter of distilled water: 28.8 g Na2HPO4 · 12H2O; 6.9 g NaH2PO4 · 2H2O; 1.4 g NH4Cl; flushed with CO2 and adjusted to pH 6.8 with 5 M NaOH), kept in a water bath at 39 °C and continuously flushed with CO2 until incubation was initiated (at least 10 min). A sample (0.8 ml) of buffered rumen fluid was acidified with 20 μl of 9 M H2SO4 and stored at −18 °C until volatile fatty acids (VFA) analysis. The stock solutions (200 μl) with BEO were added to the bottles with substrate, and equal volumes of ethanol were added to the control bottles (0 mg/l). Finally, 25 ml of buffered rumen fluid was dispensed into each bottle, and the headspace of each bottle was flushed with CO2. Bottles were sealed with butyl rubber stoppers and aluminum crimp caps, and placed in an incubator (SW 22, Julabo, Germany) at 39 °C for 24 h with shaking (frequency 90 rpm). 2.2.4. Sampling and analysis At the end of the 24 h incubation, head-space pressure of accumulated gas in the bottles was measured using a manometer (Traceable, Fisher Scientific, Pittsburgh, USA) to determine total gas production (TGP). Then, 5 ml of headspace gas was collected into a tube filled with distilled water by displacement. The limitation of this single gas sample collection after 24 h may be higher head-space pressure during fermentation that could impair microbial activity (López et al., 2007; Yáñez-Ruiz et al., 2016). According to Theodorou et al. (1994), the head-space pressure should not exceed 483 hPa, whereas López et al. (2007) found a strong correlation between gas volume and head-space pressure within the range 0–1080 hPa in fermentation bottles. Since the head-space pressure in the present experiment ranged between 418–521 hPa, it seems unlikely that the rate of fermentation was affected by pressure in the bottles. The bottles were then cooled to 4 °C to terminate fermentation, and opened to measure pH (pH 700, Eutech Instruments, Singapore), and to collect a sample of fermentation medium (0.8 ml; acidified with 20 μl of 9 M H2SO4 and stored at −18 °C) for VFA and ammonia-N analysis. Volatile fatty acids were determined according to Joch et al. (2018) using gas chromatography (GC 82 F, Labio, Czech Republic) equipped with a flame ionization detector and capillary column, and H2 was used as the carrier gas. To calculate the net production of VFA (nVFA) after 24 h of incubation, the total amount of VFA in the corresponding nonincubated sample was subtracted from the total amount of VFA produced in the incubated sample. Ammonia-N concentrations were determined by the phenol-hypochlorite reaction (Weatherburn, 1967). The remaining content of each bottle was transferred into a pre-weighed 50 ml centrifuge tube, rinsed, and centrifuged (507 × g, 10 min, 4 °C, two times). Supernatants were discarded, and precipitates were dried at 50 °C for 48 h, and weighed to determine apparent dry matter disappearance (aDMd), which was calculated as the difference between the incubated weight of the substrate and the dry weight of the fermentation residue corrected for residue weight in the blank. The concentrations of methane in the gas samples were determined using a gas chromatograph (GC 82 F, Labio, Czech Republic) equipped with a flame ionization detector and capillary column, and H2 was used as the carrier gas. A sample of 200 μl of gas was injected using a 500 μl Pressure-Lok® gastight syringe (Vici Precision Sampling, Baton Rouge, LA, USA). Calibration and calculations of the proportion of methane in the sample were carried out according to López and Newbold (2007). 178

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2.3. In vivo experiment 2.3.1. Experimental design, animals, and treatments Thirty lactating Holstein cows (two primiparous and 28 multiparous; 3 ± 1 parity) in mid-lactation (102 ± 30 DIM), with an average body weight of 629 ± 67 kg, and an average milk production of 41 ± 7 kg of milk/d, were stratified by parity, pretreatment milk production, and DIM, and then randomly assigned to one of two dietary treatments. During a 15 week period, the two treatments were: control (CON; no additive), and blend of essential oil active compounds (BEO; 1.2 g/cow/d). Weeks 1–5 served as an adaptation period, whereas weeks 6–15 were used as the experimental period. The cows were fed a total mixed ration (TMR). The basal TMR was the same for both treatment groups (Table 1), and differed only in that the BEO was mixed in the concentrate mixture of the BEO group. The cows were housed in a free-stall barn with free access to water, and were milked two times per day at 05:30 and 16:30 h. The TMR was offered ad libitum. Fresh TMR was prepared and delivered to the barn two times per day (at 04:00 h and 16:00 h), and feeding troughs were refilled with a shovel five times per day. 2.3.2. Sampling and analysis Individual TMR intake was continuously recorded using electronic balance troughs (Insentec, B.V., Marknesse, the Netherlands). Dry matter intake was calculated by adjusting daily as fed feed intake to the DM proportion of the diet. On days 7, 37, 67, and 97 of the experiment, rumen fluid samples were collected from 16 cows (n = 8 in each treatment) 4 h after morning feeding. The rumen fluid samples (˜100 ml) were collected using an oral rumen tube and a vacuum pump. The samples were immediately placed on ice and transported to the laboratory, where the pH of rumen fluid was measured. A 0.8-ml aliquot was mixed with 20 μl of 9 M H2SO4 and stored at −18 °C until analysis. The VFA and ammonia-N concentrations were determined using the Table 1 Ingredients and chemical composition (g/kg of DM unless otherwise stated) of basal TMR. Item

Amount

Ingredients Forage and liquid feed Corn silage Alfalfa silage Ensiled corn cobs with leaves (LKS) Brewers grain Wheat straw Energie MGa ProMelb Concentrate mixture Wheat Soybean meal Triticale Rapeseed meal Vitamin and mineral mixc C16d AminoPluse Sodium bicarbonate Chemical composition Dry matter, g/kg as fed Organic matter Crude protein Ether extract Starch aNDFom ADFom NFCf Net energy for lactation (MJ/kg of DM)

g/kg 597 271 161 82 74 23 17 17 403 105 61 57 56 31 22 21 4 g/kg DM 466 915 190 57 196 319 176 348 7.0

a Blend of glycerol and molasses at a 1:1 ratio (Commodity Trading, s. r. o., Olomouc, Czech Republic). b Blend of liquid protein concentrate and homogenized beet molasses in a 1:1 ratio (VVS Verměřovice, s. r. o., Verměřovice, Czech Republic). c Vitamin and mineral mix containing (per kg): 403,100 IU vitamin A, 73,494 IU vitamin D3, 1200 mg vitamin E, 133 g Ca, 33 g P, 52 g Na, 40 g Mg, 630 mg Cu, 4855 mg Mn, 3160 mg Zn, 18 mg Se, 53 mg I, 21 mg Co. d Palmitic acid (≥ 98%; LodeStar™, Berg + Schmidt Malaysia Sdn. Bhd., Selangor, Malaysia). e Bypass protein (Ag Processing Inc., Omaha, USA). f NFC (non-fiber carbohydrates) = 1000 – (aNDFom g/kg + crude protein g/kg + ether extract g/kg + ash g/kg).

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same methods as used in the in vitro experiment. Individual cow milk yields were recorded daily. Milk samples were collected from all cows on two consecutive milkings on days 7, 21, 35, 49, 63, 77, 91, and 105 of the experiment. The samples from morning and evening milkings were pooled in proportion according to individual milk yield of each milking and analyzed for milk fat, protein, lactose, and urea concentrations by infrared spectroscopy (Foss FT2, MilkoScan, Foss Electric, Hillerod, Denmark). 2.4. Statistical analysis All statistical analyses were conducted using Statistica 9.1 (StatSoft, Tulsa, OK, USA). The data from the in vitro experiment were analyzed using two-way analysis of variance, using a mixed model with BEO concentration as a fixed effect and incubation run as a random effect. Dunnett’s test was used to compare the effects of the different BEO concentrations to the control treatment (0 mg/l). Polynomial contrasts were used to determine linear (L) and/or quadratic (Q) responses to the BEO concentrations. Effects were considered significant when P < 0.05. For the data from the in vivo experiment, the effects of BEO on selected traits were evaluated using repeated measures analysis of variance. The statistical model included treatment (no additive i.e. CON, or BEO), time (day or week), and treatment × time interaction as fixed effects and cow within treatment as a random effect. Significant effects were declared at P < 0.05 and tendencies at 0.05 ≤ P < 0.10. 3. Results 3.1. Chemical composition of the blend of essential oil active compounds (BEO) Thirteen volatile compounds were identified in the BEO (Table 2), which together accounted for 255.5 mg/g of the BEO (on an as fed basis). The main essential oil compounds were thymol, m-cresol, eugenol, guaiacol, salicylic acid benzyl ester, and vanillin. None of the remaining compounds exceeded 0.040 proportion of volatile fraction (Table 2). 3.2. In vitro experiment With one exception, only the two highest concentrations of BEO (600 and 1000 mg/l) affected (P < 0.05) parameters of in vitro rumen fermentation (Table 3). The exception was the concentration of 100 mg/l, which decreased (P < 0.05) apparent dry matter disappearance (aDMd). The BEO concentration of 600 mg/l compared to control decreased (P < 0.05) methane production per DMi and relative methane production per total gas production (TGP), but molar proportions of propionate and net production of VFA (nVFA) was also decreased (P < 0.05). Furthermore, the concentration of 600 mg/l increased (P < 0.05) molar proportions of butyrate and valerate, and decreased (P < 0.05) molar proportions of iso-butyrate. The BEO concentration of 1000 mg/l compared to control increased (P < 0.05) pH and acetate:propionate ratio, while decreased (P < 0.05) TGP, molar proportions of acetate and iso-valerate, and concentrations of ammonia-N. Neither methane production per aDMd nor methane production per nVFA was affected by the addition of BEO. Inclusion of BEO at concentrations from 0 to 1000 mg/l quadratically (P < 0.001) reduced TGP, methane production per DMi, relative methane production per TGP, aDMd, nVFA, molar proportions of acetate, propionate (P = 0.011), iso-butyrate and isoTable 2 Identification and quantification of volatile active compounds in blend of essential oil compounds (mg/g of BEO) and their proportions of volatile fraction. RIb

Component

mg/g of BEO ± SD

Proportion of volatile fraction (w/w)

1031 1082 1090 1100 1145 1283 1297 1358 1395 1424 1509 1529 1857

Limonenea m-Cresol Guaiacola Linaloola Camphor Anethole Thymola Eugenola Vanillina β-Caryophyllene Butylated hydroxytoluene (BHT) Salicylic acid, pentyl ester Salicylic acid, benzyl ester Total

4.10 ± 0.11 51.50 ± 1.67 20.79 ± 0.43 8.30 ± 0.15 2.34 ± 0.08 7.00 ± 0.36 86.19 ± 2.93 22.98 ± 0.74 16.80 ± 0.59 1.71 ± 0.05 9.92 ± 0.32 4.39 ± 0.11 19.52 ± 0.60 255.54

0.016 0.202 0.081 0.033 0.009 0.027 0.337 0.090 0.066 0.007 0.039 0.017 0.076 1.000

RI, retention index; BEO, commercial blend of essential oil compounds; SD, standard deviation. a Identification confirmed by co-injection of authentic standard. b Identification based on Kovat’s retention indices (HP-5MS capillary column) and mass spectra. 180

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Table 3 Effects of blend of essential oil active compounds (BEO) on in vitro rumen fermentation parameters. Parameter

pH TGP (ml/g DMi) Methane (ml/g DMi) Methane (mmol/mol TGP) Methane (ml/g aDMd) Methane (mmol/mol nVFA) aDMd (g/g) nVFA (mmol/l) Acetate (mol/100 mol) Propionate (mol/100 mol) Butyrate (mol/100 mol) iso-butyrate (mol/100 mol) Valerate (mol/100 mol) iso-valerate (mol/100 mol) Acetate:propionate Ammonia-N (mg/100 ml)

BEO (mg/l)

SEM

0

20

100

200

600

1000

6.43 184.7 27.3 147.9 46.8 207.4 0.59 54.6 57.3 22.3 13.1 1.5 2.9 3.0 2.6 43.1

6.43 187.9 27.7 147.5 48.0 210.0 0.58 54.7 57.3 22.2 13.1 1.5 2.9 3.0 2.6 44.0

6.44 184.7 26.7 145.0 48.6 208.4 0.55* 53.3 57.4 22.1 13.1 1.5 3.0 3.0 2.6 43.8

6.45 184.2 26.6 144.6 47.5 206.5 0.56 53.5 57.1 22.2 13.3 1.5 3.0 3.0 2.6 43.2

6.45 181.9 25.7* 141.6* 47.6 207.6 0.54* 51.5* 56.6 21.6* 14.5* 1.3* 3.1* 2.9 2.7 41.2

6.47* 169.2* 22.6* 133.7* 49.4 208.1 0.46* 45.1* 53.8* 19.9* 18.5* 1.2* 3.8* 2.8* 2.8* 38.8*

0.004 1.40 0.27 1.11 0.35 1.19 0.006 0.51 0.22 0.29 0.31 0.03 0.05 0.02 0.04 0.42

P

Contrast

0.008 < 0.001 < 0.001 < 0.001 0.287 0.989 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

L

Q

0.003 < 0.001 < 0.001 < 0.001 0.128 0.887 < 0.001 < 0.001 < 0.001 0.004 < 0.001 < 0.001 < 0.001 < 0.001 0.213 < 0.001

0.013 < 0.001 < 0.001 < 0.001 0.240 0.916 < 0.001 < 0.001 < 0.001 0.011 < 0.001 < 0.001 < 0.001 < 0.001 0.434 < 0.001

BEO, blend of essential oil active compounds; SEM, standard error of the mean; P, P value (BEO concentration); L, linear contrast; Q, quadratic contrast; TGP, total gas production; DMi, dry matter incubated; aDMd, apparent dry matter disappearance; nVFA, net production of VFA. * Means within a row significantly differ from the control (0 mg/l; Dunnett’s test; P < 0.05).

valerate, and ammonia-N concentration. In contrast, pH (P = 0.013) and molar proportions of buryrate (P < 0.001) and valerate (P < 0.001) were quadratically increased (Table 3). 3.3. In vivo experiment Addition of BEO did not affect DM intake (P = 0.218). However, DM intake in the control (CON) group was numerically higher (26.2 vs. 25.0 kg/d; Table 4). Similarly, milk yield was unaffected (P = 0.102) by treatment, but in cows supplemented BEO was lower by 10.7%. The proportion of milk fat tended to be lower in the BEO group (P = 0.072). Rumen fluid of cows supplemented with BEO had a higher (P = 0.018) pH, which corresponded with lower (P = 0.006) VFA concentrations in rumen fluid of cows in the BEO group compared to cows in the CON group. Proportions of individual VFA, however, were unaffected. Table 4 Feed intake, milk production and composition, rumen fermentation parameters and body weight of Holstein cows fed diets supplemented (BEO) or not supplemented (CON) with blend of essential oil active compounds. Group Item Intake Dry matter (kg/d) Milk Yield (kg/d) Fat (g/kg) Protein (g/kg) Lactose (g/kg) MUN (mg/100 ml) Feed efficiency kg of milk/kg of DMI Rumen fluid VFA (mmol/l) Acetate (mol/100 mol) Propionate (mol/100 mol) Butyrate (mol/100 mol) Acetate:propionate Ammonia-N (mg/100 ml) pH Body weight Mean (kg) Change (kg/d)

SEM

CON

BEO

25.8

24.7

41.9 42.1 31.9 49.6 14.9

P Trt

T

Trt × T

0.60

0.218

< 0.001

0.277

37.4 37.3 32.2 49.0 14.1

1.88 0.70 0.17 0.14 0.24

0.102 0.072 0.750 0.355 0.384

< 0.001 < 0.001 0.008 0.305 < 0.001

0.430 0.590 0.904 0.386 0.200

1.69

1.53

0.06

0.094

< 0.001

0.047

125.4 65.3 19.3 14.3 3.39 12.1 6.43

113.0 66.1 18.7 14.2 3.56 11.6 6.68

2.69 0.32 0.20 0.24 0.05 0.22 0.05

0.006 0.224 0.168 0.897 0.154 0.119 0.018

0.078 0.711 0.792 0.352 0.820 0.636 0.007

0.174 0.324 0.117 0.878 0.096 0.339 0.462

646 +0.34

676 +0.57

19 0.10

0.281 0.093

< 0.001 0.001

0.014 0.490

BEO, total mixed ration supplemented with a specific blend of essential oil active compounds targeted for 1.2 g/cow/day; SEM, standard error of the mean; Trt, treatment; T, time; MUN, milk urea nitrogen. 181

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For feed efficiency (P = 0.047) and body weight (P = 0.014) treatment × time interactions were observed (Table 4, Fig. 1). Feed efficiency decreased progressively with time in BEO supplemented cows. In contrast, these cows increased body weight faster compared to CON cows. The difference became more pronounced with time. 4. Discussion Even though over the last decade several commercially available BEO have been evaluated as feed additives in ruminants, to our best knowledge, this is the first study on the use of a specific BEO that reports the analytical determined quantity of active compounds, in vitro dose-response effects and in vivo effects combined. 4.1. Chemical composition of blend of essential oil active compounds (BEO) The BEO contained approximately 256 mg of volatile active compounds per g of additive, which is in agreement with the declared content of volatile compounds. However, analyzed content of limonene and vanillin were below the declared range. In fact, analyzed content of limonene was almost 10 times lower than declared. The content of EO active compounds in BEO could be altered due to their low chemical stability and high volatility (Cobellis et al., 2016a). This emphasizes the importance of the chemical analysis of active compounds in evaluated additives. In recent years, two commercial additives with similar active compound contents were evaluated, namely Crina® Ruminants (DSM Nutritional Products, Basel, Switzerland, declared 250–400 mg/g of active compounds) (Rossi, 1995) and Agolin® Ruminant (AGOLIN SA, Bière, Switzerland, declared 200 mg/g of active compounds) (Castro-Montoya et al., 2015). Chemical analysis revealed that thymol and m-cresol were the main active components of the BEO used in this study, which together accounted for a proportion of volatile fraction of more than 0.5. The effects of thymol on rumen fermentation have been investigated in several predominantly in vitro studies, which suggest that its antimicrobial activity may be too strong and nonspecific to positively modulate fermentation in a complex microbial environment, such as the rumen (Castillejos et al., 2006; Calsamiglia et al., 2007). In contrast, to our knowledge, effects of m-cresol on rumen fermentation have not been reported in the literature. The EO effects, however, may not necessarily be attributable solely to the major components in some EO. For example, Cobellis et al. (2016b) suggested that minor EO compounds may play an important role in determining anti-methanogenic activities. Thus, some compounds less represented in the BEO could also affect rumen fermentation and/or animal production. However, these compounds usually elicit effects at relatively high concentrations. For example, eugenol at concentration of 300 mg/l increased the proportion of butyrate in rumen fermentation in vitro (Busquet et al., 2006). Patra and Yu (2014) reported that vanillin at

Fig. 1. Milk yield (a), dry matter intake (b), feed efficiency (c), and body weight (d) of Holstein cows fed diets supplemented (BEO) or not supplemented (CON) with 1.2 g/cow/d blend of essential oil active compounds. Data are shown as means with SEM of 15 cows in the CON and 15 cows in the BEO group, respectively. Vertical dashed lines indicate adaptation periods (first five weeks of the experiment). #P = 0.10. 182

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concentration of 760 mg/l decreased in vitro ammonia concentrations. Nevertheless, the effects of guaiacol were manifested at low concentration (5 mg/l), when guaiacol reduced in vitro VFA and ammonia concentrations (Castillejos et al., 2006). It is important to emphasize that both natural EO and commercial BEO are mixtures of various molecules, and therefore it is difficult to determine whether their biological effects are the result of a synergism of all molecules, or reflect only those of the main molecules present at the highest concentrations (Bakkali et al., 2008). 4.2. In vitro experiment Our results indicate that concentrations of BEO up to 200 mg/l had negligible effects on in vitro rumen fermentation, whereas the two highest concentrations (600 and 1000 mg/l) inhibited overall fermentation because nVFA production and aDMd were reduced. The concentrations of BEO 600 and 1000 mg/l also decreased methane production (ml/g DMi) by 5.7% and 17.1%, respectively. In addition, the concentration 1000 mg/l decreased ammonia-N concentration (by 10.0%). It seems, however, that these effects were only consequence of inhibition of overall fermentation. Considering the proportion of volatile active compounds in BEO (˜0.256 w/w, Table 2), this indicates that the concentration range of pure volatile compounds (mainly EO active compounds) that affects in vitro fermentation is between 150 and 250 mg/l. In keeping with this, Joch et al. (2018) found that the effective concentration of thymol, the most represented compound in BEO (Table 2), lies somewhere between 120 and 240 mg/l. The two highest concentrations of BEO (600 and 1000 mg/l) reduced the propionate proportion and elevated the butyrate proportion, which directly counteracted any positive impact on rumen fermentation (Patra and Saxena, 2010). This also suggests that the mode of action of the BEO differs from that of monensin, as monensin generally increases propionate and decreases the concentration of butyrate in the rumen (Schelling, 1984). A higher proportion of butyrate resulting from addition of higher concentrations of EO or their active compounds is well documented in vitro (Patra and Yu, 2012; Joch et al., 2018), and usually accompanies a drop in VFA concentration. Accordingly, an increase in the proportion of butyrate appears to be an indication that the concentration of EO is higher than optimal to improve rumen energy efficiency. A limited number of studies have investigated in vitro effects of commercial BEO. Moreover, in these studies, only a few low concentrations were evaluated. Pirondini et al. (2015) reported no effects of Crina® Ruminants and Agolin® Ruminant at a concentration of 16.7 mg/l on methane production in 24 h batch incubation. Likewise, Castro-Montoya et al. (2015) using similar in vitro technique found that concentrations of Agolin® Ruminant up to 30 mg/l did not affect methane production. Castillejos et al. (2007) reported weak effects of Crina® Ruminants at concentrations of 50 and 500 mg/l on rumen fermentation parameters using a continuous rumen incubation system, although, surprisingly a concentration of 5 mg/l increased VFA concentration. In our study, we failed to identify the optimal in vitro BEO concentration that would have beneficial effects without compromising the overall fermentation. Besides the negative effects on rumen fermentation, the high concentrations of BEO may have detrimental effects on overall animal health and/or function, and therefore would not be suitable for on-farm use (Yáñez-Ruiz et al., 2016). 4.3. In vivo experiment Treatment did not affect DM intake, although the average DM intake was lower in animals supplemented BEO (by approximately 4.6%). Both increases (Kung et al., 2008) and decreases (Tassoul and Shaver, 2009) in DM intake of dairy cows supplemented with 1.2 g/d of Crina® Ruminants were previously reported. These discrepancies may be a result of change in feed palatability in combination with cows’ individual taste preferences (Ginane et al., 2011), as EO active compounds have intense odors and flavors (Bakkali et al., 2008). The odor of BEO, that we subjectively perceived as unnatural and unpleasant, could be a reason for numerically lower DM intake in BEO group compared to CON group in our study. In plants, the EO may act as defensive substances because intensive odor can make the plant less attractive for herbivores (Franz and Novak, 2015). However, in our study, the feeding behavior of the cows was not analyzed to be able to assess palatability differences between the diets. The difference in milk yield was not significant between the treatment groups. However, it seems that the expected decline in milk yield due to lactation stage was more pronounced in cows in the BEO group, especially after the 5th week of the experiment (Fig. 1). Feed efficiency followed a similar pattern, i.e. feed efficiency declined over time in cows in the BEO group. The emergence of more pronounced effects as the supplementing time progressed is in agreement with Khiaosa-ard and Zebeli (2013). These authors concluded that a longer supplementation period might be necessary for some EO to affect production efficiency. Impairment of the feed efficiency could be due to a reduction in feed digestibility, as suggested by lower VFA concentration in rumen of cows in the BEO group. Importance of length of EO supplementation on rumen fermentation has been reported by Molero et al. (2004). These authors found that EO reduced degradation of crude protein in rumen after 28 days of adaptation to EO compared to unaffected protein degradation after 10 days of adaptation. The delayed effect of BEO could be connected with the ability of bacteria to counterbalance the effects of EO (Calsamiglia et al., 2007). The faster effect of BEO supplementation on rumen fermentation might be expected at higher concentrations, as suggested by our findings from the 24 h in vitro incubations. Thus, the low dose of BEO in our study might reduce feed digestibility and VFA concentration after several weeks of adaptation, which resulted in negative effects on feed efficiency. The capability of EO to adversely affect both feed digestibility and VFA production has been previously shown (Cobelis et a., 2016a). In the current study, we found a negative effect of BEO on milk fat proportion. Regarding milk production and milk fat proportion, previous studies reported varying results of supplementing cows with commercial BEO. These included, no effects of Crina Ruminants® at doses of 1 g/d (Vendramini et al., 2016) and 1.2 g/d (Tassoul and Shaver, 2009), higher milk production at a dose of 1.2 g/d of Crina® Ruminants (Kung et al., 2008), and a higher fat proportion at a dose of 1 g/d of Agolin® Ruminant (Santos et al., 2010). The BEO composition might be the key factor leading to these inconsistent results. Unfortunately, in none of the aforementioned studies were the main active components analytically quantified, which makes comparison difficult. Nevertheless, in the current study, lower milk 183

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production and a lower fat proportion in milk of cows in the BEO group compared to cows in the CON group are consistent with the ruminal data, especially with lower VFA concentrations. Lower milk yield in cows of BEO group could be related to lower availability of glucogenic VFA, in particular propionate. The level of milk production is closely related to endogenous glucose production (Hammon et al., 2010). Besides, decreased availability of lipogenic VFA, acetate and butyrate, could be responsible for lower fat proportion in the milk of BEO cows. These negative effects could be a consequence of the antimicrobial activity of BEO. The antimicrobial properties of EO and their active compounds are well documented in the literature (Dorman and Deans, 2000). It seems that supplementation of BEO inhibited rumen microbial populations. This resulted in lower feed digestibility and VFA concentrations in the rumen. Subsequently, lower availability of metabolizable energy (Van Soest, 1982) and precursors for lipid synthesis led to lower milk production and a lower proportion of milk fat, respectively. It also seems that the efficiency of nitrogen utilization was not influenced by BEO supplementation. Neither ammonia-N concentrations in the rumen nor milk urea nitrogen (MUN) and proportions of milk protein differed between groups. Conversely, in light of lower VFA concentrations in rumens of cows in the BEO group, it is surprising that in this group the average daily weight gain was almost two times higher compared to cows in the CON group (Table 4, Fig. 1). A higher body weight gain in dairy cows fed EO supplemented diets has been previously reported (Benchaar et al., 2006; Flores et al., 2013). Comparing the development of milk yield and body weight (Fig. 1), it seems to be evident that cows supplemented BEO partitioned more energy into body tissue and less into milk production compared to cows in the CON group. The shift in energy partitioning is especially pronounced between 4th and 6th week of the experiment. This shift might be regulated by insulin, which plays an important role in energy partitioning between milk and body tissue. High plasma insulin concentrations enhance glucose uptake by muscle and adipose tissue (van Knegsel et al., 2007). However, in our study, it is unlikely that concentrations of insulin were increased in BEO group since the availability of propionate (main substrate for gluconeogenesis) was reduced as suggested by lower concentrations of VFA in the rumen. Enhanced insulin sensitivity might be an explanation. Talpur et al. (2005) have shown that some EO enhanced insulin sensitivity in diabetic rats. Alternatively, metabolism of cows in BEO group might be altered by decreased concentrations of prolactin. These concentrations may be reduced by EO, as demonstrated by Sourgens et al. (1982) with thyme extract in rats. Lacasse et al. (2011) have shown a faster decline in milk production in dairy cows after five weeks of inhibition of prolactin, which coincide with the shift in energy partitioning in our study. In addition, prolactin might alter insulin sensitivity in several animal species (Tuzcu et al., 2003). However, no direct data to support these premises are available in our study. Thus, further research is necessary to elucidate effects of EO on dairy cow metabolism. 4.4. In vitro vs. In vivo The in vitro effects were manifested at concentration 600 mg/l, whereas in vivo rumen fermentation was inhibited at dose 1.2 g/ cow/d. This dose corresponds to an approximate in vitro concentration of 20 mg/l, considering an approximate rumen content volume of an adult cow of 60 l (Reynolds et al., 2004), and neglecting rumen dilution rate. It means that effects on rumen fermentation in vitro were manifested at approximately 30 times higher concentration. Moreover, this discrepancy even increases when we consider an average rumen dilution rate at 0.10 h–l (Rode et al., 1985; Reynolds et al., 2004). After that, the estimated rumen fluid flow through the rumen is around 144 l/day and consequently, the estimated rumen concentration of BEO is approximately 8.3 mg/l. It is important to emphasize, that neglecting rumen dilution rate served only for estimation of the potential maximal concentration of BEO in the rumen. Besides, the selection of dose used in in vivo experiment was not based on results of in vitro experiment, but it was selected in advance based on the manufacturer’s recommendations. In agreement with our results, Castro-Montoya et al. (2015) found that in vitro effective concentrations of Agolin® Ruminant, a BEO with different active compounds, were far greater than effective doses in vivo. Some factors may explain lower effective doses in vivo. First, unlike the cows in the in vivo experiment, there was no adaptation period in the donor cows used for in vitro incubations. Moreover, the in vitro experiment only assessed BEO effects after 24 h of incubation. However, our in vivo results suggest that effects of BEO were manifested after several weeks of supplementation. Therefore, it might be possible that especially low concentrations of BEO would demonstrate effects on rumen fermentation only after the relatively long period of supplementation. Second, the EO active compounds are sparingly soluble, thus their local concentrations may be higher, which may increase their potency in vivo. Further, impact of EO compounds may be less pronounced on microorganisms associated with liquid than on solid associated species (McIntosh et al., 2003), as rumen content is usually filtered before inoculation in vitro and the main part of solids is discarded. Also, as mentioned above, EO and/or their constituents can have multiple targets for their activity in animal bodies, and do not only affect the rumen microbial population. Unfortunately, in vitro batch incubation methods can only assess these acute effects on the rumen ecosystem. Therefore, the extrapolation of the results of in vitro studies to in vivo conditions may at times be unrepresentative (Patra and Yu, 2015), especially when evaluating multipotent bioactive compounds such as EO. 5. Conclusions Our results suggest that BEO, the additive in which EO active compounds represented approximately a quarter of its weight, had low potential to improve rumen fermentation and performance of dairy cows. No concentration of BEO improved rumen fermentation in vitro. Supplementation of BEO to dairy cows (1.2 g/cow/day) resulted in lower feed efficiency and energetic shift away from milk production to body weight gain. These negative effects might be due to an overall inhibition of rumen fermentation and at least partially due to the systemic effects of EO active compounds. However, further research is necessary to elucidate whether and how EO active compounds can affect metabolism and energy partitioning of dairy cows. This study also suggests that at low doses of EO the longer period (4–5 weeks) of supplementation might be necessary to affect dairy cow production or efficiency. This might have an 184

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implication especially for changeover experiments in which the adaptation period is usually two or three weeks. Conflict of interest There is no conflict of interest. Acknowledgements The authors wish to acknowledge the technical assistance of P. Lang. This work was supported by the Ministry of Agriculture of the Czech Republic (Project No. MZERO0717). References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/mass Spectrometry. Allured Publishing Corporation, Carol Stream, IL. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., 2008. Biological effects of essential oils – a review. Food Chem. Toxicol. 46, 446–475. https://doi.org/10.1016/j. fct.2007.09.106. Benchaar, C., Greathead, H., 2011. Essential oils and opportunities to mitigate enteric methane emissions from ruminants. Anim. Feed Sci. Technol. 166–167, 338–355. https://doi.org/10.1016/j.anifeedsci.2011.04.024. Benchaar, C., Petit, H.V., Berthiaume, R., Whyte, T.D., Chouinard, P.Y., 2006. 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