Orange (Citrus sinensis) essential oil as feed additive for sheep: nutrient intake and apparent digestibility, nitrogen balance, and rumen fermentation characteristics

Orange (Citrus sinensis) essential oil as feed additive for sheep: nutrient intake and apparent digestibility, nitrogen balance, and rumen fermentation characteristics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Orange (Citrus sinensis) essential oil as feed additive for sheep: nutrient intake and apparent digestibility, nitrogen balance, and rumen fermentation characteristics Paulo César G. Dias Junior, Ana Carolina S. Vicente, Isabela J. dos Santos, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6986736/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Tropical Animal Health and Production → Version 1 posted 4 You are reading this latest preprint version Abstract The study evaluated the effects of orange essential oil (OEO) as a feed additive on nutrient intake, digestibility, nitrogen balance, and rumen fermentation in meat sheep. Using ten rumen-cannulated Dorper × Santa Inês wethers in a 5 × 5 Latin square design, the treatments included four levels of OEO (0, 100, 500, and 1000 mg/kg DM) and a positive control (25 mg/kg DM of sodium monensin, M25). The diet consisted of a 90:10 concentrate-to-forage ratio. Increasing OEO did not influence overall nutrient intake but resulted in higher intake of DM, OM, CP, NDF, NFC, and TDN compared to M25. The apparent digestibility of NFC decreased with OEO inclusion, particularly at 1000 mg/kg DM, though it remained higher than M25. Other digestibility metrics, nitrogen absorption and nitrogen retention were unaffected. Treatments with OEO showed higher nitrogen intake than M25. Rumen fermentation characteristics, including SCFA profiles and pH, were not significantly impacted by OEO. The results indicate that OEO inclusion did not enhance nutrient digestibility, nitrogen balance, or rumen fermentation, with a minor negative effect on NFC digestibility at higher levels. additive limonene metabolism sodium monensin sheep rumen 1. Introduction Sodium monensin is the main feed additive used in ruminant diets. Its widespread use is attributed to the improvement of ruminal fermentation parameters, nutrient digestibility, and feed efficiency (Goodrich et al., 1984 ; Russel and Strobel, 1989 ; Duffield et al., 2012 ). However, due to public perception that it could potentially lead to health issues in the future, the inclusion of ionophores has been increasingly questioned (Abadi et al., 2019 ). The reason is that ionophores are considered antibiotics and may contribute to bacterial resistance to antibiotics used in human medicine (Ben et al., 2019 ; He et al., 2020 ). Given this scenario, essential oils, being a natural and sustainable alternative with antimicrobial effects, could potentially replace ionophores, as several studies have demonstrated the positive effects of different essential oils on ruminal fermentation modulation and animal performance improvement (Benchaar et al., 2008 ; Torres et al., 2020 , 2021 ; Dorantes-Iturbide et al., 2022 ). In the context of scientific research with essential oils, orange essential oil (OEO) stands out due to its high availability in the market and high concentration of D-limonene (> 90%; González-Mas et al., 2019 ). D-limonene is a monocyclic monoterpene and one of the most abundant terpenes found in nature (Arrieta et al., 2013 ). This compound is increasingly used in various industrial sectors, as well as in formulations of natural products for ruminants aimed at optimizing ruminal metabolism and animal performance (Newbold et al., 2004 ; Samii et al., 2016 ). However, studies evaluating the use of pure OEO as a source of D-limonene for ruminants are relatively scarce in the literature. Within the realm of ruminal fermentation, an in vitro study using OEO indicated an increase in propionate concentration and a reduction in methane production (Crane et al., 1957 ). Supporting the study Dorantes-Iturbide et al. ( 2022 ), it was highlighted that D-limonene improved propionate concentration in the rumen and reduced ammonia concentration without impairing microbial growth. Sallam and Abdelgaleil ( 2010 ) observed in vitro that increasing D-limonene (0, 30, 45, and 60 µL/75 mL of ruminal fluid) reduced total short-chain fatty acid (SCFA) concentration, methane production, and ruminal ammonia. In the same study, a linear reduction in dry matter (DM) and organic matter (OM) digestibility was also noted when using a diet with a concentrate/forage ratio of 50:50, which corroborates the findings of Rofiq et al. ( 2021 ), who indicated that D-limonene (100, 200, and 300 mg/kg DM) reduced methane production and in vitro neutral detergent fiber (NDF) digestibility. Conversely, Dias Junior et al. ( 2023 a) did not observe changes in total and individual SCFA concentrations when the levels of Arnica montana essential oil (61.5% D-limonene) increased (0, 450, 900, and 1350 mg/kg DM) in the diet with 90% concentrate for feedlot lambs. However, in the same study, the authors highlighted that increasing levels of Arnica montana essential oil increased crude protein digestibility, reduced ammonia nitrogen, and increased nitrogen retention. Regarding animal performance, it is noteworthy to mention the study by Kotsampasi et al., ( 2018 ), who found that OEO containing 95.2% D-limonene modulated the dry matter intake (DMI) of dairy ewes when added at a dose of 300 mg/kg DM. The authors also concluded that the dose of 450 mg/kg DM was responsible for increasing feed efficiency. (Dias Junior, Santos, et al., 2023 ), using Arnica montana essential oil containing 61.5% D-limonene, observed that increasing inclusion levels (0, 450, 900, and 1350 mg/kg DM) linearly increased DMI and average daily weight gain of lambs receiving diets with 90% concentrate. As highlighted by the previously cited studies, it is evident that OEO or D-limonene can be used as feed additives for ruminants. However, research results are still limited. In this scenario, the importance of the present study is emphasized to deeply understand the effects of OEO on ruminal metabolism and provide valuable information for the scientific community and the livestock industry. Additionally, it is important to note that most available results for this product are focused on in vitro experiments, and there are gaps regarding the effectiveness of the additive under the influence of in vivo ruminal modulation. Thus, there is a clear need for research evaluating the use of OEO through in vivo experiments. This study hypothesized that OEO would have an antimicrobial effect in the rumen and consequently modulate ruminal fermentation while increasing nutrient digestibility and nitrogen retention. This study aimed to assess the dose response of OEO and its effects on nutrient intake and apparent digestibility, nitrogen balance, and ruminal fermentation characteristics in high-concentrate diets for beef sheep. Additionally, the experiment allowed for comparisons of the effects of OEO with sodium monensin. 2. Materials and methods The experiment took place at SIPOC (Sheep and Goat Intensive Production System “Ivanete Susin”; 22º 42'24" S and 47º 37' 53" W), Brazil. All animal-related procedures followed the guidelines for Animal Research Ethics Committee of the University of São Paulo (protocol number 5160100220). 2.1. Animals, design and diets Ten rumen-cannulated Dorper × Santa Inês wethers [38.8 ± 2.58 kg initial body weight (BW); age = approximately 24 months old] were used in a replicated 5 x 5 Latin square design experiment with 5 periods of 27-d, consisting of a 21-d adaptation followed by 6-d sample collection. Wethers were housed in individual metabolic crates (1.30 × 0.55 m), designed to allow the separation and collection of urine and feces. Crates were kept in a shaded open-sided barn and the rams had ad libitum access to water during the experiment. The experimental diets consisted of five treatments: OEO0 – (negative control) inclusion of 0 mg/kg DM of OEO (CP Kelco Brasil S/A – Matão, SP, Brazil); OEO100 - inclusion of 100 mg/kg DM of OEO; OEO500 - inclusion of 500 mg/kg DM of OEO; OEO1000 - inclusion of 1000 mg/kg DM of OEO; M25 - (positive control) inclusion 25 mg/kg DM of sodium monensin (Rumensin 100, Elanco Animal Health, São Paulo, SP, Brazil). It is noteworthy to mention that sodium monensin has been used around the world, and shows consistent results, hence why it was adopted as a positive control in this study. The concentrate-to-forage [Coastcross ( Cynodon dactylon ) hay] ratio was 90:10 (DM basis) in all treatments. The experimental diets were formulated to be isoenergetic and isonitrogenous (Table 1 ) using the Small Ruminant Nutrition System (Cannas et al., 2004 ). Table 1 Ingredients and chemical composition of the total mixed ration [% of dry matter (DM)]. Item Treatments¹ OEO0 OEO100 OEO500 OEO1000 M25 Ingredients (%) Coastcross hay ( Cynodon sp ) 2 10.0 10.0 10.0 10.0 10.0 Ground corn grain 3 72.0 72.0 72.0 72.0 72.0 Soybean meal 14.0 14.0 14.0 14.0 14.0 Urea 0.4 0.4 0.4 0.4 0.4 Ammonium chloride 0.5 0.5 0.5 0.5 0.5 Limestone 1.4 1.4 1.4 1.4 1.4 Mineral premix 5 1.7 1.7 1.7 1.7 1.7 Orange essential oil 6 , mg/kg DM 0 100 500 1000 — Sodium monensin 7 , mg/kg DM — — — — 25 Chemical composition 8 (%) Dry matter 85.0 ± 0.52 85.7 ± 0.44 85.6 ± 0.55 85.4 ± 0.70 85.4 ± 0.93 Organic matter 94.5 ± 0.28 94.7 ± 0.13 94.4 ± 0.19 94.4 ± 0.11 94.1 ± 0.36 Crude protein 16.2 ± 0.52 16.3 ± 0.26 16.4 ± 0.55 16.2 ± 0.41 16.2 ± 1.17 Neutral detergent fiber 15.8 ± 1.27 15.8 ± 0.84 15.8 ± 1.02 16.0 ± 1.14 15.8 ± 1.06 Acid detergent fiber 6.3 ± 1.01 6.1 ± 0.74 6.0 ± 0.88 6.0 ± 0.79 6.2 ± 0.77 Ether extract 3.4 ± 0.47 3.4 ± 0.47 3.4 ± 0.45 3.4 ± 0.47 3.4 ± 0.56 Non-fibrous carbohydrate 58.9 ± 0.63 59.1 ± 0.33 58.7 ± 0.54 58.8 ± 0.23 57.7 ± 0.67 ME 9 , Mcal/kg of DM 2.9 2.9 2.9 2.9 2.9 1 OEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin. 2 Coastcross hay (n = 5): DM: 82.9% (± 1.16); OM: 91.3% (± 0.57); CP: 6.5% (± 0.70); NDF: 74.3% (± 2.50); ADF: 38.2% (± 1.45); EE: 1.4% (± 0.25). 3 Ground corn grain (n = 5): DM: 88.2% (± 0.76); OM: 96.6% (± 0.76); CP: 9.2% (± 0.29); NDF: 11.4% (± 0.47); ADF: 2.4% (± 0.36); EE: 3.6% (± 0.23). 4 Soybean meal (n = 5): DM: 87.9% (± 0.30); OM: 93.4% (± 0.10); CP: 45.8% (± 0.65); NDF: 11.5% (± 0.18); ADF: 6.7% (± 0.30); EE: 2.3% (± 0.21). 5 Ca: 150 g/kg; P: 40 g/kg; Na: 118 g/kg; Mg: 8 g/kg; S: 19 g/kg; Cu: 300 mg/kg; Mn: 1.250 mg/kg; Zn: 6.480 mg/kg; I: 80 mg/kg; Co: 40 mg/kg; Se: 27 mg/kg; F: 400 mg/kg. 6 Orange essential oil: 95.1% de D-limonene, 3.8% de myrcene e 1.1% de α-pinene (CP Kelco Brasil S/A – Matão, SP, Brazil). 7 Rumensin 100 (Sodium monensin, Elanco Animal Health, São Paulo, SP, Brazil). 8 Consider three samples per treatment (n = 5). 9 ME: metabolic energy, estimated using the Small Ruminant Nutrition System (Cannas et al., 2004 ). 2.2. Characterization of the compounds present in the orange essential oil The characterization of OEO was carried out in the Laboratory of Oils and Fats from the Department of Agroindustry, Food and Nutrition (LAN/ESALQ/USP), “Luiz de Queiroz'' College of Agriculture, University of São Paulo, Piracicaba, Brazil. The volatile compounds were identified by gas chromatography/mass spectrometry (HS-GC/MS) on a GCMS-QP2010 Plus (Shimadzu Corp., Tokyo, Japan). Rtx-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 µm; 5% methyl silicone as stationary phase; Bellefonte, PA) was used. The carrier gas used was helium at 1.0 mL/min flow rate. The mass spectrum was acquired via ionization at 70 eV in 40 to 500 m/z. Samples of 1 µL were injected in split mode. The temperature of the injector and detector was 220 to 230°C. The temperature ramp started at 50 ºC and was maintained for 1.5 min, followed by 200°C at 4°C/min and 240°C at 10°C/min, maintained for 7 min. The data (retention time and area on TIC) were processed for the identification of volatile compounds using the GCMS Solution software (version 4.20; Shimadzu, Tokyo, Japan). Identification was made by similarity with library data (Wiley 8.lib and FFNSC1.3.lib) and by calculation of the linear retention index (LRI) by running the C7-C30 alkane series (Supelco, Bellefonte, PA, USA). 2.3 Feed management and sampling Coastcross hay was coarsely chopped (DPM – 4 mill, Nogueira, Itapira, São Paulo, Brazil) equipped with a 10-mm pore sieve. The flint corn used herein was processed through a hammer mill (DPM – 4 mill, Nogueira, Itapira, São Paulo, Brazil). Ingredients were individually weighed using a digital scale (0.100 kg) and manually mixed. The diets were prepared with a pre-mix containing ground corn, soybean meal, ammonium chloride, limestone, and a mineral premix in a horizontal mixer with a capacity of 500 kg (Lucato, Limeira, São Paulo, Brazil). For OEO0, the chopped Coastcross hay was mixed in the feed mixer with the other ingredients. For M25, all ingredients of the OEO0 diet plus sodium monensin (Elanco Animal Health, São Paulo, SP, Brazil) were mixed in the same way as the OEO0. Twenty-five milligrams of monensin (Elanco Animal Health, São Paulo, SP, Brazil) was added per kilogram of diet (DM basis). Monensin was initially mixed with concentrate ingredients by using a mixer with a 500 kg capacity. Thereafter, chopped hay was weighed and added to the concentrate mix to produce the TMR. For OEO100, OEO500, and OEO1000 diets the orange essential oil was weighed daily on an analytical balance with an accuracy of 0.0001 g (Sartorius BA11OS, Goettingen, Germany) and then added to the pre-mix. Subsequently, chopped hay was weighed and added manually to the concentrate mix to produce the TMR, to obtain a uniform distribution of the OEO. The preparation of OEO100, OEO500, and OEO1000 was carried out every day, throughout the experimental period. Diets were offered ad libitum as total mixed ration (TMR) once a day at 7:00 h targeting 10% refusals. Samples of the feed offered of approximately 50 g were collected during five consecutive days from the 22nd to the 26th of each period and grouped by period and treatment for subsequent analysis. The waste was weighed daily to calculate the daily quantity offered based on the previous DMI. Samples of the refusals were collected during five consecutive days from the 22nd to the 26th of each period and grouped by period and treatment for subsequent chemical analysis. Samples of diets, refusals and ingredients from each period were sampled and immediately frozen at -18 ˚C. 2.4. Apparent digestibility of nutrients The intake and the apparent digestibility of nutrients were evaluated over five consecutive days from d 22 through d 26. Intakes were calculated based on DM offered after subtracting DM refused. The total amount of feces was collected in metabolic crates to estimate the apparent total digestibility of dietary nutrients. A subsample of 10% of the feces was taken after homogenization, and frozen at -18°C for later analysis. 2.5. Nitrogen balance Nitrogen balance was calculated from N in the feed consumed minus the output in feces and urine. As for feces, the urine collection was collected in metabolic crates from d 22 through d 26 at 07:00 h. The urine was collected and processed according to Dias Junior et al. ( 2023 a). For pH measurement, pH indicator strips (MQuant Merck, Darmstadt, Germany) were used. The total volume of urine was recorded daily, sampled (10%) and frozen at -18°C for later analysis. 2.6. Ruminal fluid collection On d 27, approximately 200 mL of ruminal fluid was manually obtained from each wether via ruminal cannula at 0, 4, 8, 12, 16, 20, and 24 h after feeding. Ruminal pH measurement and ruminal fluid sampling to determine SCFA and ammonia nitrogen in the rumen occurred as described by Dias Junior et al. ( 2023 a). 2.7. Laboratory analyses and calculations Feed, refusals, and fecal samples were thawed, and dried in a forced-air oven (55°C for 72 h), and ground through a 1-mm screen of a Wiley-type mill (Marconi, Piracicaba, São Paulo, Brazil) before chemical analysis. The residual moisture dry matter (DM) was removed by drying at 105°C for 24 h, in a forced air oven, and for Mineral matter (MM), Total nitrogen (N) concentration, Ether extract (EE) according to methods #930.15, #942.05, #990.03 and #920.39) respectively, of the Association of Official Analytical Chemists – AOAC ( 1990 ). The crude protein (CP) was obtained by multiplying the total N content by 6.25. Neutral detergent fiber (NDF) was determined by the sequential method according to Van Soest et al. ( 1991 ), with α-amylase and sodium sulfite. Acid detergent fiber (ADF) was determined in accordance with Goering and Van Soest ( 1970 ) with an Ankom A2000 fiber analyzer (Ankom Tech. Corp., Fairport, NY, USA). Non-fibrous carbohydrates (NFC) was calculated as: NFC (%) = 100% - (%NDF + % CP + % EE + % MM) according to NRC (2001). Total digestible nutrients (TDN) was calculated using the following equation: TDN (%) = %CP dig . + (%EE dig . × 2.25) + %NDF dig . + NFC dig . To estimate the metabolizable energy (ME), the method of Cannas et al. ( 2004 ) was used. Nutrient digestibility and N balance were calculated as described by Dias Junior et al. ( 2023 a). The SCFA concentration in ruminal fluid was determined according to Ferreira et al. ( 2016 ) and ammonia nitrogen was determined according to Broderick and Kang ( 1980 ). For statistical analysis, data were transformed to the molar ratio (mM/100 mM, i.e., the ratio between the amount of a given SCFA and the total amount observed). The total pH area under the curve and the area under pH 5.5 were calculated as described by Maulfair et al. ( 2013 ). 2.8. Statistical analysis Power analysis was conducted using the PROC POWER (SAS, 2018) to determine the number of experimental animals and samples based on DMI. The power analysis of the test considered α = 0.05 and power > 0.80. To detect minimal differences in studies that collect biological variables, a test power > 0.80 with α = 0.05 is recommended by Festing and Altman ( 2002 ) and Naseri et al. ( 2022 ). Data were analyzed using the MIXED procedure of SAS (2018). All data were subjected to the Shapiro-Wilk test for normality of residuals and removal of outliers. The homogeneity of variances by Levene's test. The statistical model for repeated measures was: Y = µ + Q m + A i (Q m ) + T j + P k + E ijk + H l + Q m + (T×H) jl + E ijklm , where µ = overall mean, Q m = Latin square fixed effect (m = 1 to 2), A i (Q m ) = animal inside Latin square effect (i = 1 to 10), T j = treatment fixed effect (j = 1 to 5), P k = period random effect (l = 1 to 5), E ijk = random residual error A, H l = fixed effect of hours after feeding, (T×H) jl = fixed effect of interaction between treatment and hours after feeding, and E ijkl = random residual error B. The covariance matrices were tested and defined according to the smallest value obtained for “Akaike's Information Criterion Corrected” (AICC). The covariance matrix that best fitted the data sets was the compound symmetric (CS). The averages for each treatment were obtained using the LSMEANS command. For the nutrient intake and apparent digestibility, and nitrogen balance, the following model was used: Y = µ + Q m + A i (Q m ) + T j + P k + E ijk , where: µ = overall mean, Q m = Latin square fixed effect (m = 1 to 2), A i (Q m ) = animal inside Latin square effect (i = 1 to 10), T j = treatment fixed effect (j = 1 to 5), P k = period random effect (l = 1 to 5), E ijk = random residual error. The effects of the inclusion levels of OEO in the diets were evaluated using linear (L) and quadratic (Q) orthogonal polynomials. The PROC IML from SAS (2018) was used to obtain the appropriate coefficients for the orthogonal contrasts. To compare the effect of the treatments, two previously defined contrasts were performed: 1 - diets with OEO vs . diet with sodium monensin (OEO vs. M25) and 2 - diet with sodium monensin vs . diet without additive (M25 vs. OEO0). The effects of the periods and the interaction between treatments and periods were defined by the F-test of the analysis of variance (ANOVA). Effects were declared significant when P < 0.05. 3. Results 3.1. Composition of orange essential oil The main volatile compounds identified in OEO are shown in Table 2 . The analysis showed that D-limonene (95.1%) is the major compound, followed by 3.8% of myrcene and 1.1% of α-pinene. Table 2 Composition of volatile compounds in orange essential oil ( Citrus sinensis ). Compound 1 % D-limonene 95.1 Myrcene 3.8 α-pinene 1.1 1 Relative amount of identified compounds based on the area of each peak in the chromatogram. 3.2. Nutrient intake and apparent digestibility Nutrient intake (DM, OM, CP, EE, NDF, ADF, NFC and TDN) was not affected when OEO was added (Table 3 ). The treatments with OEO had a higher DM, OM, CP, NDF, NFC and TDN intake than M25 ( P < 0.05). However, the intake of EE and ADF did not differ in this same contrast. There was no difference in nutrient intake between M25 and OEO0 (Table 3 ). Table 3 Effects of orange essential oil (OEO) on intake and apparent nutrient digestibility in sheep. Item 4 Treatments 1 SEM 2 P-value 3 OEO0 OEO100 OEO500 OEO1000 M25 L Q OEO vs. M25 M25 vs. OEO0 Intake (g/day) DM 855.9 974.6 866.8 843.3 756.0 52.26 0.25 0.09 0.03 0.19 OM 808.4 925.2 820.8 797.7 717.8 49.37 0.24 0.08 0.03 0.20 CP 135.5 155.1 138.4 131.8 119.4 8.27 0.20 0.09 0.03 0.18 EE 29.2 33.1 28.9 27.2 25.7 1.79 0.36 0.09 0.06 0.18 NDF 140.1 157.7 142.5 139.9 124.4 8.44 0.24 0.14 0.03 0.20 ADF 56.0 60.9 55.4 53.2 50.0 3.32 0.48 0.24 0.11 0.23 NFC 503.6 579.3 510.9 498.8 448.3 31.26 0.26 0.08 0.03 0.22 TDN 761.1 868.5 778.1 760.7 674.5 51.21 0.25 0.14 0.04 0.24 Digestibility (g/day) DM 849.8 845.2 843.1 851.5 844.5 4.39 0.42 0.81 0.70 0.42 OM 869.7 865.6 863.6 876.3 874.5 7.33 0.16 0.89 0.31 0.50 CP 861.3 858.8 856.9 862.5 868.1 9.94 0.35 0.99 0.27 0.48 EE 698.2 624.9 667.4 666.7 719.0 37.14 0.28 0.22 0.14 0.69 NDF 656.4 655.2 621.0 634.8 595.6 19.42 0.84 0.54 0.08 0.04 ADF 442.7 441.0 407.1 501.7 488.8 25.01 0.06 0.53 0.16 0.19 NFC 979.3 981.5 981.6 975.3 971.6 1.78 < 0.01 0.68 < 0.001 < 0.01 TDN 849.8 845.2 843.1 851.5 844.5 4.39 0.42 0.81 0.70 0.42 1 OEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin. 2 SEM: Standard error of the means. 3 L: linear effect; Q: quadratic effect; OEO vs. M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control vs. positive control); M25 vs. OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatments without orange essential oil (positive control vs. negative control). 4 DM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; NDF: neutral detergent fiber; ADF: acid detergent fiber; NFC: non-fibrous carbohydrate; TDN: total digestible nutrients. Increasing OEO did not affect the apparent digestibility of DM, OM, CP, EE, NDF, ADF, and TDN. However, the apparent digestibility of NFC decreased linearly when OEO was added ( P < 0.01; Table 3 ). Comparing OEO and M25 diets, the apparent digestibility of DM, OM, CP, EE, NDF, ADF, and TDN was not affected. In contrast, the apparent digestibility of NFC was higher for treatments with OEO ( P < 0.001). The apparent digestibility of DM, OM, CP, EE, ADF, and TDN was similar between M25 and OEO0. For this same comparison, the apparent digestibility of NDF and NFC was lower in the M25 ( P < 0.05). 3.3. N balance There was no effect on nitrogen (N) intake by increasing the inclusion of OEO in the diets (Table 4 ). However, treatments with OEO showed higher N intake when compared to M25 ( P = 0.03 ). Nitrogen intake between M25 and OEO0 was similar. Nitrogen excretion in feces and urine did not differ between experimental treatments. Table 4 Effects of orange essential oil (OEO) on nitrogen balance in sheep. Item 4 Treatments 1 SEM 2 P-value 3 OEO0 OEO100 OEO500 OEO1000 M25 L Q OEO vs. M25 M25 vs. OEO0 N intake, g/day 21.7 24.8 22.2 21.1 19.1 1.5 0.20 0.09 0.03 0.18 N fecal, g/day 3.1 3.4 3.2 2.9 2.6 0.1 0.12 0.08 0.23 0.35 N urine, g/day 10.8 12.9 11.2 11.0 10.9 0.9 0.51 0.10 0.42 0.93 N absorbed, g/day 18.5 21.4 18.9 18.2 16.5 1.5 0.27 0.11 0.06 0.27 N retained g/day 7.8 9.3 7.7 7.2 5.6 1.5 0.46 0.35 0.11 0.24 g/kg of N intake 338.9 348.9 332.9 305.8 253.5 40.5 0.48 0.84 0.12 0.14 g/kg of N absorbed 394.2 405.4 388.9 355.9 289.6 45.9 0.42 0.85 0.09 0.12 1 OEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin. 2 SEM: Standard error of the means. 3 L: linear effect; Q: quadratic effect; OEO vs. M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control vs. positive control); M25 vs. OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatments without orange essential oil (positive control vs. negative control). 4 N: nitrogen. The amount of N absorbed was not affected by the experimental treatments (Table 4 ). N retention (g/day, g/kg N intake and g/kg N absorption) was similar between treatments. 3.4. Ruminal fermentation characteristics Increasing OEO did not affect total SCFA concentration nor the molar concentration of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and acetate:propionate ratio (Table 5 ). There were minimal differences in ruminal fermentation variables between OEO and M25 treatments. When the M25 was compared to the OEO0, a lower total SCFA concentration was observed for M25 ( P < 0.05). In this same comparison, the individual concentration of the SCFA and the acetate:propionate ratio was not affected (Table 5 ). Table 5 Effects of orange essential oil (OEO) on ruminal fermentation characteristics in sheep. Item 4 Treatments 1 SEM 2 P-value 3 OEO0 OEO100 OEO500 OEO1000 M25 L Q OEO vs. M25 M25 vs. OEO0 H T×H SCFA total (mM) 60.6 50.1 56.1 50.1 50.4 1.07 0.85 0.06 0.66 0.05 < 0.0001 0.77 SCFA (mM/100 mM) Acetate 55.9 55.2 56.3 59.3 54.9 1.80 0.73 0.69 0.36 0.74 < 0.0001 0.26 Propionate 29.6 28.4 27.9 25.4 30.1 2.52 0.54 0.92 0.35 0.88 < 0.0001 0.33 Butyrate 11.0 11.8 11.8 12.3 11.1 1.26 0.62 0.81 0.57 0.96 0.04 0.98 Isobutyrate 0.64 0.73 0.68 0.73 0.74 0.047 0.93 0.20 0.66 0.15 < 0.0001 0.28 Valerate 1.81 2.28 2.15 1.54 2.14 0.304 0.52 0.41 0.67 0.44 0.19 0.96 Isovalerate 1.19 1.37 1.42 1.49 1.36 0.131 0.31 0.68 0.68 0.36 < 0.0001 0.35 A:P 2.23 2.45 2.73 2.80 2.26 0.271 0.16 0.94 0.22 0.93 < 0.0001 0.12 pH 5.81 5.89 5.80 5.93 5.92 0.068 0.56 0.31 0.58 0.27 < 0.0001 0.97 pH area < 5.5 (uni. pH × h/d) 49.6 49.9 51.3 44.8 34.3 9.78 0.47 0.94 0.22 0.28 - - pH area total (uni. pH × h/d) 136.9 138.5 136.9 140.4 139.9 1.66 0.58 0.41 0.52 0.22 - - Ammonia (mg/dL) 18.9 18.1 19.7 18.9 17.8 0.89 0.24 0.21 0.24 0.33 < 0.0001 0.99 1 OEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin. 2 SEM: Standard error of the means. 3 L: linear effect; Q: quadratic effect; OEO vs. M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control vs. positive control); M25 vs. OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatment without orange essential oil (positive control vs. negative control). 4 SCFA: short-chain fatty acid; A:P: acetate:propionate ratio. The experimental diets did not affect the rumen pH, the area under pH 5.5, the total area under the curve, nor the ammonia nitrogen (Table 5 ). 4. Discussion 4.1. Nutrient intake and apparent digestibility The similar DMI after increasing OEO in the diet in the current study is justified by the similarity in the chemical composition of all experimental diets coupled with a lack of effect of OEO on DM digestibility and rumen fermentation characteristics. In the present study, the expectation was that increased levels of OEO in the diet would alter rumen digestibility due to the antimicrobial effect of D-limonene. Previous studies highlighted D-limonene as a possible modulator of ruminal digestibility and fermentation (Sallam and Abdelgaleil, 2010 ; Rofiq et al., 2021 ; Dorantes-Iturbide et al., 2022 ; Dias Junior, dos Santos, et al., 2023 ). However, the results of the current study suggest that, despite the increase in OEO levels in the diet, this bioactive compound's concentration was insufficient to alter ruminal digestibility and fermentation, consequently influencing DMI. Literature indicates that the absence of effects on digestibility, ruminal fermentation, and dry matter intake may be related to the capacity of bioactive compounds present in essential oils to be absorbed in the ruminal epithelium, degraded by microbiota, or rapidly pass through the rumen (Cobellis et al., 2016 ; Torres et al., 2020 ; Dorantes-Iturbide et al., 2022 ). Thus, when absorbed or degraded, the bioavailability is altered, preventing bioactive compounds from effectively exerting their effects on the ruminal environment. Currently, the understanding of how compounds present in essential oils are absorbed or metabolized in the rumen is not completely clear, highlighting a knowledge gap that needs to be filled to guide adjustments in supplementation levels in future research exploring essential oils as nutritional additives. However, Maggiolino et al. ( 2022 ) highlighted that kids receiving D-limonene via diet (oral administration) had a higher concentration of D-limonene in the meat, indicating a high absorption rate of this compound by the ruminal epithelium. The higher DMI in rams fed with OEO compared with a treatment containing M25 can be explained by the dose of sodium monensin used in the present study. Polizel et al. ( 2021 ) reported that the dose of 24 mg/kg DM of sodium monensin reduced the DMI of lambs fed a high-energy diet, which corroborates the results of the present study using a slightly higher dose of 25 mg/kg DM sodium monensin in a diet of similar characteristics ( ie . high-concentrate inclusion). Furthermore, in the present study, it is believed that the difference in dry matter intake between lambs fed diets containing OEO and M25 may be due to a change in diet palatability, as no significant differences were found in digestibility or ruminal fermentation parameters between the additives used. Literature suggests that monensin has lower palatability for ruminants compared to essential oils (Baile et al., 1979 ; Segabinazzi et al., 2011 ). Essential oils are known to enhance diet palatability, stimulating taste and acceptability (Franz et al., 2009 ; Mucha and Witkowska, 2021 ), which may explain the increase in dry matter intake observed with the inclusion of OEO. Increasing OEO in the diet decreased the NFC digestibility. It is reported that D-limonene affects gram-negative bacteria (Gupta et al., 2021 ), which are responsible for the digestion of NFC in the rumen. Therefore, the higher inclusion of OEO in the diet proportionally increased the antimicrobial effect of the OEO, resulting in the reduction of the apparent NFC digestibility. In turn, the higher digestibility of the NFC in the treatments with OEO compared to the M25 was consistent with the fact that wether fed OEO had higher DMI (Table 3 ). There was lower NFC digestibility for M25 compared to OEO0. This effect was not expected, as similar doses of sodium monensin used in concentrate-based diets did not cause a reduction in NFC digestibility (Muntifering et al., 1981 ; Gonzalez-Momita et al., 2009 ). However, it has already been described that sodium monensin can reduce the digestibility of ruminal starch when associated with high-concentrate diets (Muntifering et al., 1981 ; Spears, 1990 ). This effect may have occurred in the present study and contributed to the lower digestibility of NFC when the wethers were fed 25 mg/kg DM of sodium monensin. The lower apparent digestibility of NDF in the M25 compared to OEO0, agrees with what is reported in the literature when sodium monensin has been included in the diet. Sodium monensin has a selective antimicrobial effect, acting on gram-positive bacteria, which are responsible for the degradation of the fibrous fraction of the diet (Russel and Strobel, 1989 ). 4.2. Nitrogen balance The higher nitrogen (N) intake observed in sheep fed with OEO compared to M25 is attributed to the increased crude protein (CP) intake in the OEO treatments. However, the lack of significant differences in N excretion via feces and urine, as well as in the amounts of N absorbed and retained, can be explained by the similarity in CP digestibility among the treatments. Crude protein digestibility plays a critical role in nitrogen metabolism, as it directly affects fecal and urinary N excretion, along with the amounts of N absorbed and retained (Broderick and Reynal, 2009 ; Mutsvangwa et al., 2016 ). Consequently, when CP digestibility remains consistent across treatments, N excretion, absorption, and retention are expected to be comparable, even in the presence of differences in CP intake. These findings underscore the importance of CP digestibility in evaluating the effects of feed additives on nitrogen metabolism and their impact on productive efficiency. 4.3. Ruminal fermentation characteristics In the present study, there were no additional positive nor deleterious effects compromising rumen fermentation in rams fed OEO1000. It is believed that the absence of effects on ruminal fermentation parameters may be associated with the possible absorption capacity by the ruminal epithelium or degradation of bioactive compounds from OEO in the rumen. In Future studies, higher inclusions of OEO is recommended to find the ideal content capable of modulating the ruminal fermentation based on the profile of the oil being utilized as suggested by Benchaar et al. ( 2008 ). Despite the latter statements, there was a reduction in total SCFA concentration for the M25 compared to OEO0 and it can be explained by the lower NDF and NFC digestibility for the M25. The lower digestibility of NDF when M25 was compared to OEO0 is in accordance with the literature, as reported when monensin is included in the diet. Monensin has a selective antimicrobial effect, acting on Gram-positive bacteria, which are responsible for the degradation of the fibrous fraction of the diet (Russel and Strobel, 1989 ). Conversely, the lower digestibility of NFC for M25 compared to OEO0 can be explained by the lower DMI observed for M25. Although this parameter did not differ, DMI was 13% lower when lambs received the M25 diet, indicating that the NFC intake per gram of DM consumed was lower for M25 compared to OEO0. It is also worth noting that monensin has been associated with a reduction in ruminal starch digestibility (Spears, 1990 ), which in the present study could negatively impact NFC digestibility, as the diets used contained corn as the main source of NFC, which was included in the same proportion in M25 and OEO0. The rumen pH was not affected by OEO supplementation. The absence of effect on the area under pH 5.5, and the total area under the pH curve indicate that the experimental treatments acted similarly on rumen pH stability. However, it is important to highlight that the inclusion of OEO up to the dose of 1000 mg/kg of DM may exert similar effects to monensin on ruminal pH, which is supported by the absence of effect on the evaluated ruminal fermentation parameters in the present study, where it was expected that diets containing OEO would have lower ruminal pH due to higher DMI compared to M25. Increases in the level of OEO supplementation did not affect the ammonia nitrogen. This result agrees with Samii et al. ( 2016 ), who observed no change in ammonia nitrogen when using different D-limonene contents in a cattle diet. Benchaar et al. ( 2008 ) and Wallace ( 2004 ) pointed out that the effects of essential oils on ammonia nitrogen were directly associated with the type of essential oil, the major compound, the dose, and the diet profile used. Thus, OEO being rich in D-limonene and associated with high-concentrate may have a less pronounced effect on modulating ammonia nitrogen. 5. Conclusion Orange essential oil did not demonstrate promising effects in modulating nutrient digestibility, nitrogen balance, and ruminal fermentation in sheep. However, there was a slight decrease in the digestibility of non-fibrous carbohydrates, particularly notable at a dose of 1000 mg/kg DM. Subsequent research should investigate the impact of orange essential oil on the rumen microbiota to understand its negative effect on non-fibrous carbohydrate digestibility, as well as assess higher levels of inclusion in the diet. Declarations Acknowledgements The authors would like to thank the São Paulo Research Foundation - Brazil (FAPESP) for funding the research, the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) for the doctoral scholarship of the first author, and the company CPKelco - Brazil for donating the orange essential oil used in this study. Author contributions Paulo César G. Dias Junior: conceptualization , methodology, investigation, validation, formal analysis, data curation, writing - original draft, Ana Carolina S. Vicente, Isabela J. dos Santos, Letícia C. B. Soares, Matheus S. P. Carlis, Adrielly L. A. da Silva, Rhaissa G. de Assis, Jamile H. Comelli: investigation, writing - review & editing; Daniel M. Polizel, Janaina S. Biava, Diogo F. A. Costa, Alexandre V. Pires: methodology, writing - review & editing, visualization; Evandro M. Ferreira: conceptualization, methodology, validation, formal analysis, writing - review & editing, supervision, funding acquisition. Funding This study was funded by the São Paulo Research Foundation – Brazil (FAPESP) under grant number 2021/01733-8. Data availability The datasets generated in the current study are available from the corresponding author on reasonable request. Ethical approval This research was submitted to Ethics Committee on the Use of Animals (CEUA) of the “Luiz de Queiroz” College of Agriculture (ESALQ), University of São Paulo (USP), Piracicaba, São Paulo, protocol number 5160100220. Declarations of interest The authors declare that they have no competing financial interests or personal relationships that could have influenced the study reported in this paper. Conflict of interest We declare no conflict of interest. References Abadi, A.T.B., Rizvanov, A.A., Haertlé, T. and Blatt, N.L., 2019. World Health Organization Report: Current Crisis of Antibiotic Resistance BioNanoScience, 9, 778–788 (BioNanoScience) AOAC, 1990. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS. Official methods of analysis, 15th ed. (Arlington) Arrieta, M.P., López, J., Ferrándiz, S. and Peltzer, M.A., 2013. 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Polizel","email":"","orcid":"","institution":"ESALQ-USP: Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"M.","lastName":"Polizel","suffix":""},{"id":494404615,"identity":"84bc8505-c438-41f3-9cf8-5bddb16e8e14","order_by":8,"name":"Janaina S. Biava","email":"","orcid":"","institution":"UNESP Campus de Botucatu: Universidade Estadual Paulista Julio de Mesquita Filho - Campus de Botucatu","correspondingAuthor":false,"prefix":"","firstName":"Janaina","middleName":"S.","lastName":"Biava","suffix":""},{"id":494404616,"identity":"73d590d0-422c-4cbd-8155-ccfeac1326aa","order_by":9,"name":"Diogo F. A. Costa","email":"","orcid":"","institution":"CQUniversity: Central Queensland University","correspondingAuthor":false,"prefix":"","firstName":"Diogo","middleName":"F. A.","lastName":"Costa","suffix":""},{"id":494404617,"identity":"42b06385-e42c-4dc7-8804-60068381832c","order_by":10,"name":"Alexandre V. Pires","email":"","orcid":"","institution":"ESALQ-USP: Universidade de Sao Paulo Escola Superior de Agricultura Luiz de Queiroz","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"V.","lastName":"Pires","suffix":""},{"id":494404618,"identity":"83374c29-97fc-46f9-80c1-0a4f2fa3cff9","order_by":11,"name":"EVANDRO MAIA FERREIRA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYLACHiBmbwCxDBgY+EE0YwMRWngOQLVINpCmBaTrAAEt/BLJzx683WPDwMPe++zDh4J78sY30p89YNxxD6cWyRlp5oZznqUx8PAcN545w6DYcNuNHHMDxjPFOLUYnDlgJs1z4DCDvUQaMzOPQQIjUAubBGNbAk4t9meOfwNr4ZF/xsz8xyDBfvOM9Gd4tRiw90Bs4ZFgY2ZmMEhI3CCRYIZXi8TxnjLJOQfSeHh40pgZewwSkmeceWNukHgGtxb+ZvZtEm8O2MjxsB9jZvjxJ8G2vx0YYh934NYCAzzIHDYGwhrQABupGkbBKBgFo2B4AwAOoU1nam0XiwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1057-5508","institution":"College of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"EVANDRO","middleName":"MAIA","lastName":"FERREIRA","suffix":""}],"badges":[],"createdAt":"2025-06-26 23:44:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6986736/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6986736/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11250-026-04969-z","type":"published","date":"2026-03-09T15:59:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":104739630,"identity":"80763014-f290-4f52-a777-3c989cc79306","added_by":"auto","created_at":"2026-03-16 16:10:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1432193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6986736/v1/f1dd22f2-83c3-4205-b4c9-6081fcf5ad9a.pdf"}],"financialInterests":"","formattedTitle":"Orange (Citrus sinensis) essential oil as feed additive for sheep: nutrient intake and apparent digestibility, nitrogen balance, and rumen fermentation characteristics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSodium monensin is the main feed additive used in ruminant diets. Its widespread use is attributed to the improvement of ruminal fermentation parameters, nutrient digestibility, and feed efficiency (Goodrich et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Russel and Strobel, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Duffield et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, due to public perception that it could potentially lead to health issues in the future, the inclusion of ionophores has been increasingly questioned (Abadi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The reason is that ionophores are considered antibiotics and may contribute to bacterial resistance to antibiotics used in human medicine (Ben et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given this scenario, essential oils, being a natural and sustainable alternative with antimicrobial effects, could potentially replace ionophores, as several studies have demonstrated the positive effects of different essential oils on ruminal fermentation modulation and animal performance improvement (Benchaar et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Torres et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dorantes-Iturbide et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the context of scientific research with essential oils, orange essential oil (OEO) stands out due to its high availability in the market and high concentration of D-limonene (\u0026gt;\u0026thinsp;90%; Gonz\u0026aacute;lez-Mas et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). D-limonene is a monocyclic monoterpene and one of the most abundant terpenes found in nature (Arrieta et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This compound is increasingly used in various industrial sectors, as well as in formulations of natural products for ruminants aimed at optimizing ruminal metabolism and animal performance (Newbold et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Samii et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, studies evaluating the use of pure OEO as a source of D-limonene for ruminants are relatively scarce in the literature.\u003c/p\u003e\u003cp\u003eWithin the realm of ruminal fermentation, an \u003cem\u003ein vitro\u003c/em\u003e study using OEO indicated an increase in propionate concentration and a reduction in methane production (Crane et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). Supporting the study Dorantes-Iturbide et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), it was highlighted that D-limonene improved propionate concentration in the rumen and reduced ammonia concentration without impairing microbial growth. Sallam and Abdelgaleil (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) observed \u003cem\u003ein vitro\u003c/em\u003e that increasing D-limonene (0, 30, 45, and 60 \u0026micro;L/75 mL of ruminal fluid) reduced total short-chain fatty acid (SCFA) concentration, methane production, and ruminal ammonia. In the same study, a linear reduction in dry matter (DM) and organic matter (OM) digestibility was also noted when using a diet with a concentrate/forage ratio of 50:50, which corroborates the findings of Rofiq et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who indicated that D-limonene (100, 200, and 300 mg/kg DM) reduced methane production and \u003cem\u003ein vitro\u003c/em\u003e neutral detergent fiber (NDF) digestibility. Conversely, Dias Junior et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea) did not observe changes in total and individual SCFA concentrations when the levels of \u003cem\u003eArnica montana\u003c/em\u003e essential oil (61.5% D-limonene) increased (0, 450, 900, and 1350 mg/kg DM) in the diet with 90% concentrate for feedlot lambs. However, in the same study, the authors highlighted that increasing levels of \u003cem\u003eArnica montana\u003c/em\u003e essential oil increased crude protein digestibility, reduced ammonia nitrogen, and increased nitrogen retention.\u003c/p\u003e\u003cp\u003eRegarding animal performance, it is noteworthy to mention the study by Kotsampasi et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who found that OEO containing 95.2% D-limonene modulated the dry matter intake (DMI) of dairy ewes when added at a dose of 300 mg/kg DM. The authors also concluded that the dose of 450 mg/kg DM was responsible for increasing feed efficiency. (Dias Junior, Santos, et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), using \u003cem\u003eArnica montana\u003c/em\u003e essential oil containing 61.5% D-limonene, observed that increasing inclusion levels (0, 450, 900, and 1350 mg/kg DM) linearly increased DMI and average daily weight gain of lambs receiving diets with 90% concentrate.\u003c/p\u003e\u003cp\u003eAs highlighted by the previously cited studies, it is evident that OEO or D-limonene can be used as feed additives for ruminants. However, research results are still limited. In this scenario, the importance of the present study is emphasized to deeply understand the effects of OEO on ruminal metabolism and provide valuable information for the scientific community and the livestock industry. Additionally, it is important to note that most available results for this product are focused on \u003cem\u003ein vitro\u003c/em\u003e experiments, and there are gaps regarding the effectiveness of the additive under the influence of \u003cem\u003ein vivo\u003c/em\u003e ruminal modulation. Thus, there is a clear need for research evaluating the use of OEO through \u003cem\u003ein vivo\u003c/em\u003e experiments. This study hypothesized that OEO would have an antimicrobial effect in the rumen and consequently modulate ruminal fermentation while increasing nutrient digestibility and nitrogen retention.\u003c/p\u003e\u003cp\u003eThis study aimed to assess the dose response of OEO and its effects on nutrient intake and apparent digestibility, nitrogen balance, and ruminal fermentation characteristics in high-concentrate diets for beef sheep. Additionally, the experiment allowed for comparisons of the effects of OEO with sodium monensin.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003eThe experiment took place at SIPOC (Sheep and Goat Intensive Production System \u0026ldquo;Ivanete Susin\u0026rdquo;; 22\u0026ordm; 42'24\" S and 47\u0026ordm; 37' 53\" W), Brazil. All animal-related procedures followed the guidelines for Animal Research Ethics Committee of the University of S\u0026atilde;o Paulo (protocol number 5160100220).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals, design and diets\u003c/h2\u003e\u003cp\u003eTen rumen-cannulated Dorper \u0026times; Santa In\u0026ecirc;s wethers [38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.58 kg initial body weight (BW); age\u0026thinsp;=\u0026thinsp;approximately 24 months old] were used in a replicated 5 x 5 Latin square design experiment with 5 periods of 27-d, consisting of a 21-d adaptation followed by 6-d sample collection. Wethers were housed in individual metabolic crates (1.30 \u0026times; 0.55 m), designed to allow the separation and collection of urine and feces. Crates were kept in a shaded open-sided barn and the rams had \u003cem\u003ead libitum\u003c/em\u003e access to water during the experiment.\u003c/p\u003e\u003cp\u003eThe experimental diets consisted of five treatments: \u003cb\u003eOEO0\u003c/b\u003e \u0026ndash; (negative control) inclusion of 0 mg/kg DM of OEO (CP Kelco Brasil S/A \u0026ndash; Mat\u0026atilde;o, SP, Brazil); \u003cb\u003eOEO100\u003c/b\u003e - inclusion of 100 mg/kg DM of OEO; \u003cb\u003eOEO500\u003c/b\u003e - inclusion of 500 mg/kg DM of OEO; \u003cb\u003eOEO1000\u003c/b\u003e - inclusion of 1000 mg/kg DM of OEO; \u003cb\u003eM25\u003c/b\u003e - (positive control) inclusion 25 mg/kg DM of sodium monensin (Rumensin 100, Elanco Animal Health, S\u0026atilde;o Paulo, SP, Brazil). It is noteworthy to mention that sodium monensin has been used around the world, and shows consistent results, hence why it was adopted as a positive control in this study. The concentrate-to-forage [Coastcross (\u003cem\u003eCynodon dactylon\u003c/em\u003e) hay] ratio was 90:10 (DM basis) in all treatments. The experimental diets were formulated to be isoenergetic and isonitrogenous (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using the Small Ruminant Nutrition System (Cannas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIngredients and chemical composition of the total mixed ration [% of dry matter (DM)].\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItem\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eTreatments\u0026sup1;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOEO0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOEO100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOEO500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOEO1000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM25\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIngredients (%)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCoastcross hay (\u003cem\u003eCynodon sp\u003c/em\u003e)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGround corn grain\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e72.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e72.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e72.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUrea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmmonium chloride\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLimestone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMineral premix\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrange essential oil\u003csup\u003e6\u003c/sup\u003e, mg/kg DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium monensin\u003csup\u003e7\u003c/sup\u003e, mg/kg DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eChemical composition\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e \u003cem\u003e(%)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDry matter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e85.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e85.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e85.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganic matter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e94.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e94.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e94.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeutral detergent fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcid detergent fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEther extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNon-fibrous carbohydrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e59.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e58.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e57.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eME\u003csup\u003e9\u003c/sup\u003e, Mcal/kg of DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003eOEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e2\u003c/sup\u003eCoastcross hay (n\u0026thinsp;=\u0026thinsp;5): DM: 82.9% (\u0026plusmn;\u0026thinsp;1.16); OM: 91.3% (\u0026plusmn;\u0026thinsp;0.57); CP: 6.5% (\u0026plusmn;\u0026thinsp;0.70); NDF: 74.3% (\u0026plusmn;\u0026thinsp;2.50); ADF: 38.2% (\u0026plusmn;\u0026thinsp;1.45); EE: 1.4% (\u0026plusmn;\u0026thinsp;0.25).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e3\u003c/sup\u003eGround corn grain (n\u0026thinsp;=\u0026thinsp;5): DM: 88.2% (\u0026plusmn;\u0026thinsp;0.76); OM: 96.6% (\u0026plusmn;\u0026thinsp;0.76); CP: 9.2% (\u0026plusmn;\u0026thinsp;0.29); NDF: 11.4% (\u0026plusmn;\u0026thinsp;0.47); ADF: 2.4% (\u0026plusmn;\u0026thinsp;0.36); EE: 3.6% (\u0026plusmn;\u0026thinsp;0.23).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e4\u003c/sup\u003eSoybean meal (n\u0026thinsp;=\u0026thinsp;5): DM: 87.9% (\u0026plusmn;\u0026thinsp;0.30); OM: 93.4% (\u0026plusmn;\u0026thinsp;0.10); CP: 45.8% (\u0026plusmn;\u0026thinsp;0.65); NDF: 11.5% (\u0026plusmn;\u0026thinsp;0.18); ADF: 6.7% (\u0026plusmn;\u0026thinsp;0.30); EE: 2.3% (\u0026plusmn;\u0026thinsp;0.21).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e5\u003c/sup\u003e Ca: 150 g/kg; P: 40 g/kg; Na: 118 g/kg; Mg: 8 g/kg; S: 19 g/kg; Cu: 300 mg/kg; Mn: 1.250 mg/kg; Zn: 6.480 mg/kg; I: 80 mg/kg; Co: 40 mg/kg; Se: 27 mg/kg; F: 400 mg/kg.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e6\u003c/sup\u003eOrange essential oil: 95.1% de D-limonene, 3.8% de myrcene e 1.1% de α-pinene (CP Kelco Brasil S/A \u0026ndash; Mat\u0026atilde;o, SP, Brazil).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e7\u003c/sup\u003eRumensin 100 (Sodium monensin, Elanco Animal Health, S\u0026atilde;o Paulo, SP, Brazil).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e8\u003c/sup\u003eConsider three samples per treatment (n\u0026thinsp;=\u0026thinsp;5).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e9\u003c/sup\u003eME: metabolic energy, estimated using the \u003cem\u003eSmall Ruminant Nutrition System\u003c/em\u003e (Cannas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Characterization of the compounds present in the orange essential oil\u003c/h2\u003e\u003cp\u003eThe characterization of OEO was carried out in the Laboratory of Oils and Fats from the Department of Agroindustry, Food and Nutrition (LAN/ESALQ/USP), \u0026ldquo;Luiz de Queiroz'' College of Agriculture, University of S\u0026atilde;o Paulo, Piracicaba, Brazil.\u003c/p\u003e\u003cp\u003eThe volatile compounds were identified by gas chromatography/mass spectrometry (HS-GC/MS) on a GCMS-QP2010 Plus (Shimadzu Corp., Tokyo, Japan). Rtx-5MS fused silica capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m; 5% methyl silicone as stationary phase; Bellefonte, PA) was used. The carrier gas used was helium at 1.0 mL/min flow rate. The mass spectrum was acquired via ionization at 70 eV in 40 to 500 m/z. Samples of 1 \u0026micro;L were injected in split mode. The temperature of the injector and detector was 220 to 230\u0026deg;C. The temperature ramp started at 50 \u0026ordm;C and was maintained for 1.5 min, followed by 200\u0026deg;C at 4\u0026deg;C/min and 240\u0026deg;C at 10\u0026deg;C/min, maintained for 7 min. The data (retention time and area on TIC) were processed for the identification of volatile compounds using the GCMS Solution software (version 4.20; Shimadzu, Tokyo, Japan). Identification was made by similarity with library data (Wiley 8.lib and FFNSC1.3.lib) and by calculation of the linear retention index (LRI) by running the C7-C30 alkane series (Supelco, Bellefonte, PA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Feed management and sampling\u003c/h2\u003e\u003cp\u003eCoastcross hay was coarsely chopped (DPM \u0026ndash; 4 mill, Nogueira, Itapira, S\u0026atilde;o Paulo, Brazil) equipped with a 10-mm pore sieve. The flint corn used herein was processed through a hammer mill (DPM \u0026ndash; 4 mill, Nogueira, Itapira, S\u0026atilde;o Paulo, Brazil). Ingredients were individually weighed using a digital scale (0.100 kg) and manually mixed.\u003c/p\u003e\u003cp\u003eThe diets were prepared with a pre-mix containing ground corn, soybean meal, ammonium chloride, limestone, and a mineral premix in a horizontal mixer with a capacity of 500 kg (Lucato, Limeira, S\u0026atilde;o Paulo, Brazil). For OEO0, the chopped Coastcross hay was mixed in the feed mixer with the other ingredients. For M25, all ingredients of the OEO0 diet plus sodium monensin (Elanco Animal Health, S\u0026atilde;o Paulo, SP, Brazil) were mixed in the same way as the OEO0. Twenty-five milligrams of monensin (Elanco Animal Health, S\u0026atilde;o Paulo, SP, Brazil) was added per kilogram of diet (DM basis). Monensin was initially mixed with concentrate ingredients by using a mixer with a 500 kg capacity. Thereafter, chopped hay was weighed and added to the concentrate mix to produce the TMR.\u003c/p\u003e\u003cp\u003eFor OEO100, OEO500, and OEO1000 diets the orange essential oil was weighed daily on an analytical balance with an accuracy of 0.0001 g (Sartorius BA11OS, Goettingen, Germany) and then added to the pre-mix. Subsequently, chopped hay was weighed and added manually to the concentrate mix to produce the TMR, to obtain a uniform distribution of the OEO. The preparation of OEO100, OEO500, and OEO1000 was carried out every day, throughout the experimental period.\u003c/p\u003e\u003cp\u003eDiets were offered ad libitum as total mixed ration (TMR) once a day at 7:00 h targeting 10% refusals. Samples of the feed offered of approximately 50 g were collected during five consecutive days from the 22nd to the 26th of each period and grouped by period and treatment for subsequent analysis. The waste was weighed daily to calculate the daily quantity offered based on the previous DMI. Samples of the refusals were collected during five consecutive days from the 22nd to the 26th of each period and grouped by period and treatment for subsequent chemical analysis. Samples of diets, refusals and ingredients from each period were sampled and immediately frozen at -18 ˚C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Apparent digestibility of nutrients\u003c/h2\u003e\u003cp\u003eThe intake and the apparent digestibility of nutrients were evaluated over five consecutive days from d 22 through d 26. Intakes were calculated based on DM offered after subtracting DM refused. The total amount of feces was collected in metabolic crates to estimate the apparent total digestibility of dietary nutrients. A subsample of 10% of the feces was taken after homogenization, and frozen at -18\u0026deg;C for later analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Nitrogen balance\u003c/h2\u003e\u003cp\u003eNitrogen balance was calculated from N in the feed consumed minus the output in feces and urine. As for feces, the urine collection was collected in metabolic crates from d 22 through d 26 at 07:00 h. The urine was collected and processed according to Dias Junior et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea). For pH measurement, pH indicator strips (MQuant Merck, Darmstadt, Germany) were used. The total volume of urine was recorded daily, sampled (10%) and frozen at -18\u0026deg;C for later analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Ruminal fluid collection\u003c/h2\u003e\u003cp\u003eOn d 27, approximately 200 mL of ruminal fluid was manually obtained from each wether via ruminal cannula at 0, 4, 8, 12, 16, 20, and 24 h after feeding. Ruminal pH measurement and ruminal fluid sampling to determine SCFA and ammonia nitrogen in the rumen occurred as described by Dias Junior et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Laboratory analyses and calculations\u003c/h2\u003e\u003cp\u003eFeed, refusals, and fecal samples were thawed, and dried in a forced-air oven (55\u0026deg;C for 72 h), and ground through a 1-mm screen of a Wiley-type mill (Marconi, Piracicaba, S\u0026atilde;o Paulo, Brazil) before chemical analysis. The residual moisture dry matter (DM) was removed by drying at 105\u0026deg;C for 24 h, in a forced air oven, and for Mineral matter (MM), Total nitrogen (N) concentration, Ether extract (EE) according to methods #930.15, #942.05, #990.03 and #920.39) respectively, of the Association of Official Analytical Chemists \u0026ndash; AOAC (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). The crude protein (CP) was obtained by multiplying the total N content by 6.25. Neutral detergent fiber (NDF) was determined by the sequential method according to Van Soest et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), with α-amylase and sodium sulfite. Acid detergent fiber (ADF) was determined in accordance with Goering and Van Soest (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) with an Ankom A2000 fiber analyzer (Ankom Tech. Corp., Fairport, NY, USA).\u003c/p\u003e\u003cp\u003eNon-fibrous carbohydrates (NFC) was calculated as: NFC (%)\u0026thinsp;=\u0026thinsp;100% - (%NDF + % CP + % EE + % MM) according to NRC (2001). Total digestible nutrients (TDN) was calculated using the following equation: TDN (%) = %CP\u003csub\u003edig\u003c/sub\u003e. + (%EE\u003csub\u003edig\u003c/sub\u003e. \u0026times; 2.25) + %NDF\u003csub\u003edig\u003c/sub\u003e. + NFC\u003csub\u003edig\u003c/sub\u003e. To estimate the metabolizable energy (ME), the method of Cannas et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) was used. Nutrient digestibility and N balance were calculated as described by Dias Junior et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe SCFA concentration in ruminal fluid was determined according to Ferreira et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and ammonia nitrogen was determined according to Broderick and Kang (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). For statistical analysis, data were transformed to the molar ratio (mM/100 mM, i.e., the ratio between the amount of a given SCFA and the total amount observed). The total pH area under the curve and the area under pH 5.5 were calculated as described by Maulfair et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e\u003cp\u003ePower analysis was conducted using the PROC POWER (SAS, 2018) to determine the number of experimental animals and samples based on DMI. The power analysis of the test considered α\u0026thinsp;=\u0026thinsp;0.05 and power\u0026thinsp;\u0026gt;\u0026thinsp;0.80. To detect minimal differences in studies that collect biological variables, a test power\u0026thinsp;\u0026gt;\u0026thinsp;0.80 with α\u0026thinsp;=\u0026thinsp;0.05 is recommended by Festing and Altman (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and Naseri et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eData were analyzed using the MIXED procedure of SAS (2018). All data were subjected to the Shapiro-Wilk test for normality of residuals and removal of outliers. The homogeneity of variances by Levene's test. The statistical model for repeated measures was: Y\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;Q\u003csub\u003em\u003c/sub\u003e + A\u003csub\u003ei\u003c/sub\u003e(Q\u003csub\u003em\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;T\u003csub\u003ej\u003c/sub\u003e + P\u003csub\u003ek\u003c/sub\u003e + E\u003csub\u003eijk\u003c/sub\u003e + H\u003csub\u003el\u003c/sub\u003e + Q\u003csub\u003em\u003c/sub\u003e + (T\u0026times;H)\u003csub\u003ejl\u003c/sub\u003e + E\u003csub\u003eijklm\u003c/sub\u003e, where \u0026micro;\u0026thinsp;=\u0026thinsp;overall mean, Q\u003csub\u003em\u003c/sub\u003e = Latin square fixed effect (m\u0026thinsp;=\u0026thinsp;1 to 2), A\u003csub\u003ei\u003c/sub\u003e(Q\u003csub\u003em\u003c/sub\u003e )\u0026thinsp;=\u0026thinsp;animal inside Latin square effect (i\u0026thinsp;=\u0026thinsp;1 to 10), T\u003csub\u003ej\u003c/sub\u003e = treatment fixed effect (j\u0026thinsp;=\u0026thinsp;1 to 5), P\u003csub\u003ek\u003c/sub\u003e = period random effect (l\u0026thinsp;=\u0026thinsp;1 to 5), E\u003csub\u003eijk\u003c/sub\u003e = random residual error A, H\u003csub\u003el\u003c/sub\u003e = fixed effect of hours after feeding, (T\u0026times;H)\u003csub\u003ejl\u003c/sub\u003e = fixed effect of interaction between treatment and hours after feeding, and E\u003csub\u003eijkl\u003c/sub\u003e = random residual error B. The covariance matrices were tested and defined according to the smallest value obtained for \u0026ldquo;Akaike's Information Criterion Corrected\u0026rdquo; (AICC). The covariance matrix that best fitted the data sets was the compound symmetric (CS). The averages for each treatment were obtained using the LSMEANS command. For the nutrient intake and apparent digestibility, and nitrogen balance, the following model was used: Y\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;Q\u003csub\u003em\u003c/sub\u003e + A\u003csub\u003ei\u003c/sub\u003e(Q\u003csub\u003em\u003c/sub\u003e)\u0026thinsp;+\u0026thinsp;T\u003csub\u003ej\u003c/sub\u003e + P\u003csub\u003ek\u003c/sub\u003e + E\u003csub\u003eijk\u003c/sub\u003e, where: \u0026micro;\u0026thinsp;=\u0026thinsp;overall mean, Q\u003csub\u003em\u003c/sub\u003e = Latin square fixed effect (m\u0026thinsp;=\u0026thinsp;1 to 2), A\u003csub\u003ei\u003c/sub\u003e (Q\u003csub\u003em\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;animal inside Latin square effect (i\u0026thinsp;=\u0026thinsp;1 to 10), T\u003csub\u003ej\u003c/sub\u003e = treatment fixed effect (j\u0026thinsp;=\u0026thinsp;1 to 5), P\u003csub\u003ek\u003c/sub\u003e = period random effect (l\u0026thinsp;=\u0026thinsp;1 to 5), E\u003csub\u003eijk\u003c/sub\u003e = random residual error. The effects of the inclusion levels of OEO in the diets were evaluated using linear (L) and quadratic (Q) orthogonal polynomials. The PROC IML from SAS (2018) was used to obtain the appropriate coefficients for the orthogonal contrasts. To compare the effect of the treatments, two previously defined contrasts were performed: 1 - diets with OEO \u003cem\u003evs\u003c/em\u003e. diet with sodium monensin (OEO \u003cem\u003evs.\u003c/em\u003e M25) and 2 - diet with sodium monensin \u003cem\u003evs\u003c/em\u003e. diet without additive (M25 \u003cem\u003evs.\u003c/em\u003e OEO0). The effects of the periods and the interaction between treatments and periods were defined by the F-test of the analysis of variance (ANOVA). Effects were declared significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Composition of orange essential oil\u003c/h2\u003e\u003cp\u003eThe main volatile compounds identified in OEO are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The analysis showed that D-limonene (95.1%) is the major compound, followed by 3.8% of myrcene and 1.1% of α-pinene.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComposition of volatile compounds in orange essential oil (\u003cem\u003eCitrus sinensis\u003c/em\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompound\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-limonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e95.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMyrcene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eα-pinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003csup\u003e1\u003c/sup\u003eRelative amount of identified compounds based on the area of each peak in the chromatogram.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Nutrient intake and apparent digestibility\u003c/h2\u003e\u003cp\u003eNutrient intake (DM, OM, CP, EE, NDF, ADF, NFC and TDN) was not affected when OEO was added (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The treatments with OEO had a higher DM, OM, CP, NDF, NFC and TDN intake than M25 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the intake of EE and ADF did not differ in this same contrast. There was no difference in nutrient intake between M25 and OEO0 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of orange essential oil (OEO) on intake and apparent nutrient digestibility in sheep.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItem\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eTreatments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOEO0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOEO100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOEO500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOEO1000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eQ\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eOEO \u003cem\u003evs.\u003c/em\u003e M25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eM25 \u003cem\u003evs.\u003c/em\u003e OEO0\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIntake\u003c/em\u003e (g/day)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e855.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e974.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e866.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e843.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e756.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e52.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e808.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e925.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e820.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e797.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e717.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e49.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e135.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e155.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e138.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e131.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e119.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e25.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e140.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e157.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e142.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e139.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e124.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e53.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e503.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e579.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e510.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e498.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e448.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e31.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e761.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e868.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e778.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e760.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e674.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e51.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eDigestibility\u003c/em\u003e (g/day)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e849.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e845.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e843.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e851.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e844.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e869.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e865.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e863.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e876.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e874.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e861.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e858.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c3\"\u003e\u003cp\u003e624.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e667.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e666.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e719.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e37.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e656.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e655.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e621.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e634.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e595.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e442.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e441.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e407.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e501.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e488.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e25.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e979.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e981.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e981.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e975.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e971.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e849.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e845.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e843.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e851.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e844.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e1\u003c/sup\u003eOEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e2\u003c/sup\u003eSEM: Standard error of the means.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e3\u003c/sup\u003eL: linear effect; Q: quadratic effect; OEO \u003cem\u003evs.\u003c/em\u003e M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control \u003cem\u003evs.\u003c/em\u003e positive control); M25 \u003cem\u003evs.\u003c/em\u003e OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatments without orange essential oil (positive control \u003cem\u003evs.\u003c/em\u003e negative control).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e4\u003c/sup\u003eDM: dry matter; OM: organic matter; CP: crude protein; EE: ether extract; NDF: neutral detergent fiber; ADF: acid detergent fiber; NFC: non-fibrous carbohydrate; TDN: total digestible nutrients.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIncreasing OEO did not affect the apparent digestibility of DM, OM, CP, EE, NDF, ADF, and TDN. However, the apparent digestibility of NFC decreased linearly when OEO was added (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Comparing OEO and M25 diets, the apparent digestibility of DM, OM, CP, EE, NDF, ADF, and TDN was not affected. In contrast, the apparent digestibility of NFC was higher for treatments with OEO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The apparent digestibility of DM, OM, CP, EE, ADF, and TDN was similar between M25 and OEO0. For this same comparison, the apparent digestibility of NDF and NFC was lower in the M25 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3. N balance\u003c/h2\u003e\u003cp\u003eThere was no effect on nitrogen (N) intake by increasing the inclusion of OEO in the diets (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, treatments with OEO showed higher N intake when compared to M25 (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.03\u003c/em\u003e). Nitrogen intake between M25 and OEO0 was similar. Nitrogen excretion in feces and urine did not differ between experimental treatments.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of orange essential oil (OEO) on nitrogen balance in sheep.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItem\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eTreatments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOEO0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOEO100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOEO500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOEO1000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eQ\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eOEO \u003cem\u003evs.\u003c/em\u003e M25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eM25 \u003cem\u003evs.\u003c/em\u003e OEO0\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN intake, g/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e22.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e21.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN fecal, g/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN urine, g/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e11.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN absorbed, g/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e18.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e16.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN retained\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg/kg of N intake\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e338.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e348.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e332.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e305.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e253.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e40.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eg/kg of N absorbed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e394.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e405.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e388.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e355.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e289.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e45.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e1\u003c/sup\u003eOEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e2\u003c/sup\u003eSEM: Standard error of the means.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e3\u003c/sup\u003eL: linear effect; Q: quadratic effect; OEO \u003cem\u003evs.\u003c/em\u003e M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control \u003cem\u003evs.\u003c/em\u003e positive control); M25 \u003cem\u003evs.\u003c/em\u003e OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatments without orange essential oil (positive control \u003cem\u003evs.\u003c/em\u003e negative control).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003e4\u003c/sup\u003eN: nitrogen.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe amount of N absorbed was not affected by the experimental treatments (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). N retention (g/day, g/kg N intake and g/kg N absorption) was similar between treatments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Ruminal fermentation characteristics\u003c/h2\u003e\u003cp\u003eIncreasing OEO did not affect total SCFA concentration nor the molar concentration of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and acetate:propionate ratio (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). There were minimal differences in ruminal fermentation variables between OEO and M25 treatments. When the M25 was compared to the OEO0, a lower total SCFA concentration was observed for M25 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In this same comparison, the individual concentration of the SCFA and the acetate:propionate ratio was not affected (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of orange essential oil (OEO) on ruminal fermentation characteristics in sheep.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"13\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eItem\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eTreatments\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSEM\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c13\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOEO0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOEO100\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOEO500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOEO1000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eM25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eQ\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eOEO \u003cem\u003evs.\u003c/em\u003e M25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eM25 \u003cem\u003evs.\u003c/em\u003e OEO0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eT\u0026times;H\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSCFA total (mM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e50.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"13\" nameend=\"c13\" namest=\"c1\"\u003e\u003cp\u003eSCFA (mM/100 mM)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e59.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e54.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePropionate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e29.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e30.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButyrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsobutyrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.304\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsovalerate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA:P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.271\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.068\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH area\u0026thinsp;\u0026lt;\u0026thinsp;5.5 (uni. pH \u0026times; h/d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e49.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e34.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH area total (uni. pH \u0026times; h/d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e136.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e138.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e136.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e140.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e139.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmmonia (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e1\u003c/sup\u003eOEO0: inclusion of 0 mg/kg DM of orange essential oil; OEO100: inclusion of 100 mg/kg DM of orange essential oil; OEO500: inclusion of 500 mg/kg DM of orange essential oil; OEO1000: inclusion of 1000 mg/kg DM of orange essential oil; M25: inclusion of 25 mg/kg DM of sodium monensin.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e2\u003c/sup\u003eSEM: Standard error of the means.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e3\u003c/sup\u003eL: linear effect; Q: quadratic effect; OEO \u003cem\u003evs.\u003c/em\u003e M25: treatments containing orange essential oil vs. treatment with 25 mg/kg DM of sodium monensin (negative control \u003cem\u003evs.\u003c/em\u003e positive control); M25 \u003cem\u003evs.\u003c/em\u003e OEO0: treatment with 25 mg/kg DM of sodium monensin vs. treatment without orange essential oil (positive control \u003cem\u003evs.\u003c/em\u003e negative control).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003e4\u003c/sup\u003eSCFA: short-chain fatty acid; A:P: acetate:propionate ratio.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe experimental diets did not affect the rumen pH, the area under pH 5.5, the total area under the curve, nor the ammonia nitrogen (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Nutrient intake and apparent digestibility\u003c/h2\u003e\u003cp\u003eThe similar DMI after increasing OEO in the diet in the current study is justified by the similarity in the chemical composition of all experimental diets coupled with a lack of effect of OEO on DM digestibility and rumen fermentation characteristics. In the present study, the expectation was that increased levels of OEO in the diet would alter rumen digestibility due to the antimicrobial effect of D-limonene. Previous studies highlighted D-limonene as a possible modulator of ruminal digestibility and fermentation (Sallam and Abdelgaleil, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rofiq et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Dorantes-Iturbide et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dias Junior, dos Santos, et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the results of the current study suggest that, despite the increase in OEO levels in the diet, this bioactive compound's concentration was insufficient to alter ruminal digestibility and fermentation, consequently influencing DMI. Literature indicates that the absence of effects on digestibility, ruminal fermentation, and dry matter intake may be related to the capacity of bioactive compounds present in essential oils to be absorbed in the ruminal epithelium, degraded by microbiota, or rapidly pass through the rumen (Cobellis et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Torres et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dorantes-Iturbide et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, when absorbed or degraded, the bioavailability is altered, preventing bioactive compounds from effectively exerting their effects on the ruminal environment. Currently, the understanding of how compounds present in essential oils are absorbed or metabolized in the rumen is not completely clear, highlighting a knowledge gap that needs to be filled to guide adjustments in supplementation levels in future research exploring essential oils as nutritional additives. However, Maggiolino et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) highlighted that kids receiving D-limonene via diet (oral administration) had a higher concentration of D-limonene in the meat, indicating a high absorption rate of this compound by the ruminal epithelium.\u003c/p\u003e\u003cp\u003eThe higher DMI in rams fed with OEO compared with a treatment containing M25 can be explained by the dose of sodium monensin used in the present study. Polizel et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that the dose of 24 mg/kg DM of sodium monensin reduced the DMI of lambs fed a high-energy diet, which corroborates the results of the present study using a slightly higher dose of 25 mg/kg DM sodium monensin in a diet of similar characteristics (\u003cem\u003eie\u003c/em\u003e. high-concentrate inclusion). Furthermore, in the present study, it is believed that the difference in dry matter intake between lambs fed diets containing OEO and M25 may be due to a change in diet palatability, as no significant differences were found in digestibility or ruminal fermentation parameters between the additives used. Literature suggests that monensin has lower palatability for ruminants compared to essential oils (Baile et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Segabinazzi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Essential oils are known to enhance diet palatability, stimulating taste and acceptability (Franz et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mucha and Witkowska, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which may explain the increase in dry matter intake observed with the inclusion of OEO.\u003c/p\u003e\u003cp\u003eIncreasing OEO in the diet decreased the NFC digestibility. It is reported that D-limonene affects gram-negative bacteria (Gupta et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which are responsible for the digestion of NFC in the rumen. Therefore, the higher inclusion of OEO in the diet proportionally increased the antimicrobial effect of the OEO, resulting in the reduction of the apparent NFC digestibility. In turn, the higher digestibility of the NFC in the treatments with OEO compared to the M25 was consistent with the fact that wether fed OEO had higher DMI (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There was lower NFC digestibility for M25 compared to OEO0. This effect was not expected, as similar doses of sodium monensin used in concentrate-based diets did not cause a reduction in NFC digestibility (Muntifering et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Gonzalez-Momita et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, it has already been described that sodium monensin can reduce the digestibility of ruminal starch when associated with high-concentrate diets (Muntifering et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Spears, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). This effect may have occurred in the present study and contributed to the lower digestibility of NFC when the wethers were fed 25 mg/kg DM of sodium monensin.\u003c/p\u003e\u003cp\u003eThe lower apparent digestibility of NDF in the M25 compared to OEO0, agrees with what is reported in the literature when sodium monensin has been included in the diet. Sodium monensin has a selective antimicrobial effect, acting on gram-positive bacteria, which are responsible for the degradation of the fibrous fraction of the diet (Russel and Strobel, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Nitrogen balance\u003c/h2\u003e\u003cp\u003eThe higher nitrogen (N) intake observed in sheep fed with OEO compared to M25 is attributed to the increased crude protein (CP) intake in the OEO treatments. However, the lack of significant differences in N excretion via feces and urine, as well as in the amounts of N absorbed and retained, can be explained by the similarity in CP digestibility among the treatments.\u003c/p\u003e\u003cp\u003eCrude protein digestibility plays a critical role in nitrogen metabolism, as it directly affects fecal and urinary N excretion, along with the amounts of N absorbed and retained (Broderick and Reynal, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mutsvangwa et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Consequently, when CP digestibility remains consistent across treatments, N excretion, absorption, and retention are expected to be comparable, even in the presence of differences in CP intake. These findings underscore the importance of CP digestibility in evaluating the effects of feed additives on nitrogen metabolism and their impact on productive efficiency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Ruminal fermentation characteristics\u003c/h2\u003e\u003cp\u003eIn the present study, there were no additional positive nor deleterious effects compromising rumen fermentation in rams fed OEO1000. It is believed that the absence of effects on ruminal fermentation parameters may be associated with the possible absorption capacity by the ruminal epithelium or degradation of bioactive compounds from OEO in the rumen. In Future studies, higher inclusions of OEO is recommended to find the ideal content capable of modulating the ruminal fermentation based on the profile of the oil being utilized as suggested by Benchaar et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the latter statements, there was a reduction in total SCFA concentration for the M25 compared to OEO0 and it can be explained by the lower NDF and NFC digestibility for the M25. The lower digestibility of NDF when M25 was compared to OEO0 is in accordance with the literature, as reported when monensin is included in the diet. Monensin has a selective antimicrobial effect, acting on Gram-positive bacteria, which are responsible for the degradation of the fibrous fraction of the diet (Russel and Strobel, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Conversely, the lower digestibility of NFC for M25 compared to OEO0 can be explained by the lower DMI observed for M25. Although this parameter did not differ, DMI was 13% lower when lambs received the M25 diet, indicating that the NFC intake per gram of DM consumed was lower for M25 compared to OEO0. It is also worth noting that monensin has been associated with a reduction in ruminal starch digestibility (Spears, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), which in the present study could negatively impact NFC digestibility, as the diets used contained corn as the main source of NFC, which was included in the same proportion in M25 and OEO0.\u003c/p\u003e\u003cp\u003eThe rumen pH was not affected by OEO supplementation. The absence of effect on the area under pH 5.5, and the total area under the pH curve indicate that the experimental treatments acted similarly on rumen pH stability. However, it is important to highlight that the inclusion of OEO up to the dose of 1000 mg/kg of DM may exert similar effects to monensin on ruminal pH, which is supported by the absence of effect on the evaluated ruminal fermentation parameters in the present study, where it was expected that diets containing OEO would have lower ruminal pH due to higher DMI compared to M25.\u003c/p\u003e\u003cp\u003eIncreases in the level of OEO supplementation did not affect the ammonia nitrogen. This result agrees with Samii et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who observed no change in ammonia nitrogen when using different D-limonene contents in a cattle diet. Benchaar et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and Wallace (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) pointed out that the effects of essential oils on ammonia nitrogen were directly associated with the type of essential oil, the major compound, the dose, and the diet profile used. Thus, OEO being rich in D-limonene and associated with high-concentrate may have a less pronounced effect on modulating ammonia nitrogen.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOrange essential oil did not demonstrate promising effects in modulating nutrient digestibility, nitrogen balance, and ruminal fermentation in sheep. However, there was a slight decrease in the digestibility of non-fibrous carbohydrates, particularly notable at a dose of 1000 mg/kg DM. Subsequent research should investigate the impact of orange essential oil on the rumen microbiota to understand its negative effect on non-fibrous carbohydrate digestibility, as well as assess higher levels of inclusion in the diet.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the S\u0026atilde;o Paulo Research Foundation - Brazil (FAPESP) for funding the research, the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) for the doctoral scholarship of the first author, and the company CPKelco - Brazil for donating the orange essential oil used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePaulo C\u0026eacute;sar G. Dias Junior:\u0026nbsp;\u003c/strong\u003econceptualization\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003emethodology, investigation, validation, formal analysis, data curation, writing - original draft, \u003cstrong\u003eAna Carolina S. Vicente, Isabela J. dos Santos, Let\u0026iacute;cia C. B. Soares, Matheus S. P. Carlis, Adrielly L. A. da Silva, Rhaissa G. de Assis, Jamile H. Comelli:\u0026nbsp;\u003c/strong\u003einvestigation, writing - review \u0026amp; editing; \u003cstrong\u003eDaniel M. Polizel, Janaina S. Biava, Diogo F. A. Costa, Alexandre V. Pires:\u0026nbsp;\u003c/strong\u003emethodology,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewriting - review \u0026amp; editing, visualization; \u003cstrong\u003eEvandro M. Ferreira:\u003c/strong\u003e conceptualization, methodology, validation, formal analysis, writing - review \u0026amp; editing, supervision, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the S\u0026atilde;o Paulo Research Foundation \u0026ndash; Brazil (FAPESP) under grant number 2021/01733-8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was submitted to Ethics Committee on the Use of Animals (CEUA) of the \u0026ldquo;Luiz de Queiroz\u0026rdquo; College of Agriculture (ESALQ), University of S\u0026atilde;o Paulo (USP), Piracicaba, S\u0026atilde;o Paulo, protocol number 5160100220.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have influenced the study reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbadi, A.T.B., Rizvanov, A.A., Haertl\u0026eacute;, T. and Blatt, N.L., 2019. World Health Organization Report: Current Crisis of Antibiotic Resistance BioNanoScience, 9, 778\u0026ndash;788 (BioNanoScience)\u003c/li\u003e\n \u003cli\u003eAOAC, 1990. ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS. Official methods of analysis, 15th ed. (Arlington)\u003c/li\u003e\n \u003cli\u003eArrieta, M.P., L\u0026oacute;pez, J., Ferr\u0026aacute;ndiz, S. and Peltzer, M.A., 2013. Characterization of PLA-limonene blends for food packaging Characterization of PLA-limonene blends for food packaging, 32, 760\u0026ndash;768\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBaile, C.A., McLaughlin, C.L., Potter, E.L. and Chalupa, W., 1979. Feeding behavior changes of cattle during introduction of monensin with roughage or concentrate diets. Journal of animal science, 48, 1501\u0026ndash;1508\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBen, Y., Fu, C., Hu, M., Liu, L., Wong, M.H. and Zheng, C., 2019. Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: A review Environmental Research, 169, 483\u0026ndash;493\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBenchaar, C., Calsamiglia, S., Chaves, A. V., Fraser, G.R., Colombatto, D., McAllister, T.A. and Beauchemin, K.A., 2008. A review of plant-derived essential oils in ruminant nutrition and production Animal Feed Science and Technology, 145, 209\u0026ndash;228\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBroderick, G.A. and Kang, J.H., 1980. Automated simultaneous determination of dmmonia and total amino acids in ruminal fluid and in vitro media Journal of Dairy Science, 63, 64\u0026ndash;75\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBroderick, G.A. and Reynal, S.M., 2009. Effect of source of rumen-degraded protein on production and ruminal metabolism in lactating dairy cows Journal of Dairy Science, 92, 2822\u0026ndash;2834 (Elsevier)\u003c/li\u003e\n \u003cli\u003eCannas, A., Tedeschi, L.O., Fox, D.G., Pell, A.N. and Van Soest, P.J., 2004. A mechanistic model for predicting the nutrient requirements and feed biological values for sheep Journal of Animal Science, 82, 149\u0026ndash;169\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCobellis, G., Trabalza-Marinucci, M. and Yu, Z., 2016. Critical evaluation of essential oils as rumen modifiers in ruminant nutrition: A review Science of The Total Environment, 545\u0026ndash;546, 556\u0026ndash;568\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCrane, A., Nelson, W.O. and Brown, R.E., 1957. Effects of D-Limonene and \u0026alpha;-D-Pinene on in Vitro Carbohydrate Dissimilation and Methane Formation by Rumen Bacteria Journal of Dairy Science, 40, 1317\u0026ndash;1323\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDias Junior, P.C.G., dos Santos, I.J., Gasparina, J.M., Bandoria, N.A., Soares, L.C.B., da Silva, A.L.A., de Assis, R.G., Polizel, D.M., Biava, J.S., Pires, A.V. and Ferreira, E.M., 2023. Essential oil from Arnica montana alters the protein metabolism in lambs fed with high-concentrate diets Small Ruminant Research, 219\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDias Junior, P.C.G., Santos, I.J., Silva, A.L.A., Assis, R.G., Vicente, A.C.S., Carlis, M.S.P., Soares, L.C.B., Comelli, J.H., Biava, J.S., Araujo, R.C., Pires, A.V. and Ferreira, E.M., 2023. 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Meta-analysis of the effects of monensin in beef cattle on feed efficiency, body weight gain, and dry matter intake Journal of Animal Science, 90, 4583\u0026ndash;4592\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFerreira, E.M., Pires, A.V., Susin, I., Biehl, M.V., Gentil, R.S., Parente, M. de O.M., Polizel, D.M., Vaz Di Mambro Ribeiro, C. and de Almeida, E., 2016. Nutrient digestibility and ruminal fatty acid metabolism in lambs supplemented with soybean oil partially replaced by fish oil blend Animal Feed Science and Technology, 216, 30\u0026ndash;39 (Elsevier B.V.)\u003c/li\u003e\n \u003cli\u003eFesting, M.F.W. and Altman, D.G., 2002. Guidelines for the design and statistical analysis of experiments using laboratory animals ILAR Journal, 43, 244\u0026ndash;257\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFranz, C., Baser, K.H.C. and Windish, W., 2009. Essential oils and aromatic plants in animal feeding \u0026ndash; a European perspective. A review Flavour and Fragrance Journal, 25, 327\u0026ndash;340\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGoering, H.K. and Van Soest, P.J., 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures and Some Applications), (US Department of Agriculture: Washington, DC)\u003c/li\u003e\n \u003cli\u003eGonz\u0026aacute;lez-Mas, M.C., Rambla, J.L., L\u0026oacute;pez-Gresa, M.P., Amparo Bl\u0026aacute;zquez, M. and Granell, A., 2019. Volatile compounds in citrus essential oils: A comprehensive review Frontiers in Plant Science, 10, 1\u0026ndash;18\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGonzalez-Momita, M.L., Kawas, J.R., Garc\u0026iacute;a-Castillo, R., Gonzalez-Morteo, C., Aguirre-Ortega, J., Hernandez-Vidal, G., Fimbres-Durazo, H., Pic\u0026oacute;n-Rubio, F.J. and Lu, C.D., 2009. Nutrient intake, digestibility, mastication and ruminal fermentation of Pelibuey lambs fed finishing diets with ionophore (monensin or lasalocid) and sodium malate Small Ruminant Research, 83, 1\u0026ndash;6\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGoodrich, R.D., Garrett, J.. E., Gast, D.R., Kirick, M.A., Larson, D.A. and Meiske, J.. C., 1984. Influence of Monensin on the Performance of Cattle Journal of Animal Science, 58, 1484\u0026ndash;1498\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGupta, A., Jeyakumar, E. and Lawrence, R., 2021. Journey of limonene as an antimicrobial agent Journal of Pure and Applied Microbiology, 15, 1094\u0026ndash;1110\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHe, Y., Yuan, Q., Mathieu, J., Stadler, L., Senehi, N., Sun, R. and Alvarez, P.J.J., 2020. Antibiotic resistance genes from livestock waste: occurrence, dissemination, and treatment npj Clean Water, 3, 1\u0026ndash;11\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKotsampasi, B., Tsiplakou, E., Christodoulou, C., Mavrommatis, A., Mitsiopoulou, C., Karaiskou, C., Sossidou, E., Fragioudakis, N., Kapsomenos, I., Bampidis, V.A., Christodoulou, V. and Zervas, G., 2018. Effects of dietary orange peel essential oil supplementation on milk yield and composition, and blood and milk antioxidant status of dairy ewes Animal Feed Science and Technology, 245, 20\u0026ndash;31\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMaggiolino, A., Faccia, M., Homen, B.W.B., Hopkins, D.L., Bragaglio, A., Natrella, G., Mazzone, A. and Palo, P., 2022. The effect of oral or respiratory exposure to limonene on goat kid performance and meat quality Meat Science, 191\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMaulfair, D.D., McIntyre, K.K. and Heinrichs, A.J., 2013. Subacute ruminal acidosis and total mixed ration preference in lactating dairy cows1 Journal of Dairy Science, 96, 6610\u0026ndash;6620 (Elsevier)\u003c/li\u003e\n \u003cli\u003eMucha, W. and Witkowska, D., 2021. The applicability of essential oils in different stages of production of animal-based foods Molecules, 26\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMuntifering, R.B., Theurer, B. and Noon, T.H., 1981. Effects of monensin on site and extent of whole corn digestion and bacterial protein synthesis in beef steers. Journal of animal science, 53, 1565\u0026ndash;1573\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMutsvangwa, T., Davies, K.L., McKinnon, J.J. and Christensen, D.A., 2016. 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Low doses of monensin for lambs fed diets containing high level of ground flint corn Scientia Agricola, 78, 1\u0026ndash;8\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRofiq, M.N., Negara, W., Martono, S., Gopar, R.A. and Boga, M., 2021. Potential effect of some essential oils on rumen methane reduction and digestibility by in Vitro incubation technique IOP Conference Series: Earth and Environmental Science, 905\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRussel, J.B. and Strobel, H.J., 1989. Effect of lonophores on ruminal fermentation Applied and Environmental Microbiology, 55, 1\u0026ndash;6\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSallam, S.M.A. and Abdelgaleil, S.A.M., 2010. Efecto de diferentes niveles de aceite esencial de c\u0026iacute;trico y su componente activo en la fermentaci\u0026oacute;n microbiana ruminal y la producci\u0026oacute;n in vitro de metano Revista Cubana de Ciencia Agricola, 44, 373\u0026ndash;378\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSamii, S.S., Wallace, N., Nagaraja, T.G., Engstrom, M.A., Miesner, M.D., Armendariz, C.K. and Titgemeyer, E.C., 2016. Effects of limonene on ruminal fusobacterium necrophorum concentrations, fermentation, and lysine degradation in cattle Journal of Animal Science, 94, 3420\u0026ndash;3430\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSegabinazzi, L.R., Vi\u0026eacute;gas, J., Freitas, L.S., Brondani, I.L., Argenta, F.M. and Binotto, J., 2011. Behavior patterns of cows with Charolais or Nellore breed predominance fed diets with plant extract or monensin sodium Revista Brasileira de Zootecnia, 40, 2954\u0026ndash;2962\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSpears, J.W., 1990. lonophores and Nutrient Digestion and Absorption in Ruminants The Journal of Nutrition, 120, 632\u0026ndash;638\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTorres, R.N.S., Moura, D.C., Ghedini, C.P., Ezequiel, J.M.B. and Almeida, M.T.C., 2020. Meta-analysis of the effects of essential oils on ruminal fermentation and performance of sheep Small Ruminant Research, 189, 106148 (Elsevier)\u003c/li\u003e\n \u003cli\u003eTorres, R.N.S., Paschoaloto, J.R., Ezequiel, J.M.B., da Silva, D.A.V. and Almeida, M.T.C., 2021. Meta-analysis of the effects of essential oil as an alternative to monensin in diets for beef cattle Veterinary Journal, 272, 105659 (Elsevier Ltd.)\u003c/li\u003e\n \u003cli\u003eVan Soest, P.J., Robertson, J.B. and Lewis, B.A., 1991. 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Proceedings of the Nutrition Society, 63, 621\u0026ndash;629 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"additive, limonene, metabolism, sodium monensin, sheep, rumen","lastPublishedDoi":"10.21203/rs.3.rs-6986736/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6986736/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe study evaluated the effects of orange essential oil (OEO) as a feed additive on nutrient intake, digestibility, nitrogen balance, and rumen fermentation in meat sheep. Using ten rumen-cannulated Dorper \u0026times; Santa In\u0026ecirc;s wethers in a 5 \u0026times; 5 Latin square design, the treatments included four levels of OEO (0, 100, 500, and 1000 mg/kg DM) and a positive control (25 mg/kg DM of sodium monensin, M25). The diet consisted of a 90:10 concentrate-to-forage ratio. Increasing OEO did not influence overall nutrient intake but resulted in higher intake of DM, OM, CP, NDF, NFC, and TDN compared to M25. The apparent digestibility of NFC decreased with OEO inclusion, particularly at 1000 mg/kg DM, though it remained higher than M25. Other digestibility metrics, nitrogen absorption and nitrogen retention were unaffected. Treatments with OEO showed higher nitrogen intake than M25. Rumen fermentation characteristics, including SCFA profiles and pH, were not significantly impacted by OEO. The results indicate that OEO inclusion did not enhance nutrient digestibility, nitrogen balance, or rumen fermentation, with a minor negative effect on NFC digestibility at higher levels.\u003c/p\u003e","manuscriptTitle":"Orange (Citrus sinensis) essential oil as feed additive for sheep: nutrient intake and apparent digestibility, nitrogen balance, and rumen fermentation characteristics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 13:54:07","doi":"10.21203/rs.3.rs-6986736/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-08T12:29:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T15:39:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-27T06:10:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Animal Health and Production","date":"2025-06-26T19:43:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-animal-health-and-production","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trop","sideBox":"Learn more about [Tropical Animal Health and Production](https://www.springer.com/journal/11250)","snPcode":"11250","submissionUrl":"https://submission.nature.com/new-submission/11250/3","title":"Tropical Animal Health and Production","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2d14d24c-0935-40d1-a232-d38ec5a31135","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:05:49+00:00","versionOfRecord":{"articleIdentity":"rs-6986736","link":"https://doi.org/10.1007/s11250-026-04969-z","journal":{"identity":"tropical-animal-health-and-production","isVorOnly":false,"title":"Tropical Animal Health and Production"},"publishedOn":"2026-03-09 15:59:57","publishedOnDateReadable":"March 9th, 2026"},"versionCreatedAt":"2025-08-12 13:54:07","video":"","vorDoi":"10.1007/s11250-026-04969-z","vorDoiUrl":"https://doi.org/10.1007/s11250-026-04969-z","workflowStages":[]},"version":"v1","identity":"rs-6986736","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6986736","identity":"rs-6986736","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}