A Blend of Essential Oils (Blend of Eugenol, Linalool, Anethole, and Cinnamaldehyde) Increases Ruminal Propionate and Improves Total Tract Starch Digestibility in Steers Fed a Dry-Rolled Corn-Based Finishing Diet

Abstract

Feed additives based on essential oils (EOs) have emerged as a potential alternative to ionophores for diets with elevated grain inclusion. Also, on some occasions, EOs have been used in combination with monensin, with variable results. A metabolism trial was conducted using a 2 × 2 factorial arrangement of treatments, evaluating supplementation with (A) monensin sodium (0 mg/steer daily vs. 400 mg. steer daily) and (B) a blend of EOs (eugenol, linalool, anethole, and cinnamaldehyde, 0 g/d vs. 14 g/d). Four Red Angus steers (BW = 435 ± 9.0 kg) with ruminal and duodenal cannulas were used, and the study was conducted as a Latin square with four periods of 28 days each. Ruminal fermentation and nutrient digestibility at different levels (ruminal, intestinal, and total tract) were determined. The EOs increased total tract starch digestibility (p = 0.05) by 4.5% and propionate concentration (p = 0.03) by 30.9%. Furthermore, EOs decreased acetate (p = 0.04) by 7.4% as well as the acetate to propionate ratio (p = 0.03). In conclusion, our results suggest that it is safe to combine this EO blend with monensin for feedlot diets. The EO blend improved starch digestibility and increased efficiency of the ruminal fermentation end-products, which suggests it could be beneficial in diets based upon grain.

1. Introduction

Modern feedlot diets are characterized by high inclusion levels of concentrates to meet the energy requirements and support the elevated rates of gain of beef cattle [1]. In the rumen, fermentation of glucose can follow different pathways, resulting in various end-products, with the main ones being short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate, and the hydroxy acid lactate, among others [2]. From a productive standpoint, the most desirable end-product is propionate, since this SCFA captures all carbons present in the glucose molecule, as opposed to acetate, which only captures two thirds of the carbons present [1,2]. Additionally, lactate is an undesirable fermentation end-product that, due to its low acid dissociation constant (pKa = 3.86), can lead to ruminal acidosis [3,4]. Diets with high concentrate inclusion typically contain elevated levels of starch and limited amounts of physically effective fiber, favoring the accumulation of lactate, which is the main cause of ruminal acidosis [3,4].

The ionophore monensin sodium is widely used in feedlot diets to ameliorate the effects of ruminal acidosis, decrease the acetate to propionate ratio (A:P), improve the feed conversion rate, and enhance the growth performance of cattle [1,5,6]. However, the possibility of emerging bacterial resistance has triggered vigorous controversy regarding the use of antibiotics, such as monensin, as growth promoters [7]. In addition, changes in consumer preferences and governmental policies have driven a shift towards natural products, creating a demand for alternatives to ionophores [8,9,10].

Essential oils (EOs) are phytogenic compounds extracted from plants that have multiple uses as biotechnological tools in agriculture, health, food sciences, and more [10,11,12,13]. Some EOs have the potential to modulate rumen fermentation [11,14], in particular under high-grain diets where previous studies have shown reductions in the A:P ratio and ammonia nitrogen concentration [11,14]. Such impacts on ruminal fermentation make them potential candidates as feed additives to improve feed efficiency utilization and increase growth performance; however, this premise has been tested and the results are variable [15,16], highlighting the need to evaluate each blend of EOs independently of previous trials. On some occasions, the combination of certain EOs with monensin has been reported to be antagonistic, with deleterious effects on intake and growth performance [16].

A recent meta-analysis evaluated the effects of the substitution of monensin by EOs [17]. This study determined that, on average, cattle supplemented with EOs exhibit greater intake, less ruminal ammonia nitrogen, larger ribeye area, and greater dressing percentage, as compared to monensin-supplemented cattle [17]. However, on average, supplementation with EOs was less effective in preventing the development of liver abscesses, which are less prevalent in monensin-fed cattle [17]. Even though, since there are many EOs available as feed additives for cattle, this premise should not be generalized, and each blend should be evaluated in combination with ionophores. Furthermore, the effects of EOs are dependent on the supplemented dose and the ruminal pH [11,18,19].

Eugenol is a phenolic compound that exhibits wide-ranging antibacterial efficacy, targeting both Gram-negative and Gram-positive organisms [11,20]. It is known to modulate ruminal fermentation by reducing acetate and ammonia nitrogen and increasing propionate [18,19,21]. Yet it can decrease total VFAs at high doses [11,18,19]. In a previous trial with finishing steers, supplementation with eugenol tended to increase ruminal propionate and tended to reduce the acetate to propionate ratio [20]. Anethol, a phenylpropanoid, also has antimicrobial activity, and it has been shown to modulate ruminal fermentation [11]. Some of the known ruminal effects of anethole are a decrease in the concentrations of ammonia nitrogen and acetate, and an increase in propionate concentrations [22,23]. Cinnamaldehyde is a phenylpropanoid with antimicrobial activity [11,24] and has been proven to inhibit protein degradation, and depending on the dose, it can alter VFA proportions in favor of propionate [11,25]. Furthermore, supplementation with cinnamaldehyde to feedlot steers has shown to stimulate intake during the initial feeding period, decreasing blood urea nitrogen and non-esterified fatty acids [24]. Linalool, a monoterpene alcohol, has been proven to have potent antioxidant and immune modulation effects [26,27].

However, it is not clear whether monensin and this particular EO blend would interact, and if this EO blend can potentially replace monensin in finishing diets. Moreover, it is unknown how this product and its combination with monensin will affect ruminal, post-ruminal, and total tract nutrient digestibility and utilization, and ruminal fermentation parameters. Thus, there is a critical need to evaluate the effects of this EO blend as a substitute for monensin, and/or their combination, on beef cattle nutrient utilization and ruminal fermentation.

2. Materials and Methods

All procedures involving animal care and handling were conducted under the approval of the South Dakota State University Institutional Animal Care and Use Committee (protocol #2308-073E).

2.1. Animals, Experimental Design, and Dietary Treatments

Four Red Angus steers (BW = 435 ± 9.0 kg) with ruminal and duodenal fistulae were used in a 4 × 4 Latin Square design with a 2 × 2 factorial arrangement of treatments. This design resulted in 4 biological replicates per treatment and a total of 16 individual observations across the 4 experimental periods. The factors evaluated were as follows: Factor (A): monensin sodium supplementation: 0 mg/d (MON−) vs. 400 mg/d (MON+); Factor (B): blend of EOs supplementation: 0 g/d (EO−) vs. 14 g/d (EO+). The commercial EO additive utilized in this study consisted of a proprietary blend of eugenol, linalool, anethole, and cinnamaldehyde. The combination of factors resulted in four treatments:

(1)

EO−MON−; 0 g/d EO and 0 mg/d MON.

(2)

EO+MON−; 14 g/d EO and 0 mg/d MON.

(3)

EO−MON+; 0 g/d EO and 400 mg/d MON.

(4)

EO+MON+; 14 g/d EO and 400 mg/d MON.

The trial was conducted during four 28 d periods, consisting of a 14 d diet adaptation followed by a 14 d collection period. Steers were housed individually with ad libitum access to feed and water and were fed once daily at 0700h throughout the trial. Feed was offered to allow for a minimum of 5–10% carry-over feed to ensure ad libitum intake.

The basal diet fed to the steers consisted of dry rolled corn, grass hay, DDGS, and vitamin and mineral premix, and presented a 10:90 roughage-to-concentrate ratio on a DM basis (

Table 1. Proportion of ingredients and chemical composition of the basal diet.

Item	%
Ingredients, % DM
Dry rolled corn	67.0
Grass hay	10.0
Dried distillers’ grains plus solubles	20.0
Vitamin and mineral premix 1	3.0
Chemical composition, % DM
Organic matter	94.99
Crude protein	13.92
Ether extract	3.21
Neutral detergent fiber	31.44
Starch	44.02

¹ Made up of (DM basis) corn carrier (0.6%), urea (0.6%), CaCO₃ (1.5%), and vitamin/trace mineral supplement (0.3%; 650,000 IU/g Vitamin A, 500 IU/g Vitamin E, 900 ppm Zn, and 300 ppm Cu).

). Diets were mixed weekly, and representative samples of individual feed ingredients were collected during each of the four mixing events per period and pooled by period for chemical analysis. Feed additives for the MON+ and EO+ treatments were dosed daily at 0700h in the rumen in gelatin capsules to guarantee the steers received the targeted dose.

2.2. Nutrient Intake and Digestibility

Feed intake and apparent total tract digestibility were determined over five consecutive days (d 16 to d 20 of each period). Feeds offered were weighed daily, sampled, and stored at −20 °C from days 16 to 20 of each experimental period, while orts were weighed daily, sampled, and stored at −20 °C from days 17 to 21. All samples were partially dried in a forced-air ventilation oven at 60 °C for 72 h. Once dried, the samples were ground to a 1 mm particle size using a bench-top cutting mill (Thomas Scientific Wiley Mill, Model 4), pooled per animal and period, and stored for further analysis.

Total fecal collections were performed over five consecutive days (days 16 to 20) in each experimental period. Briefly, steers were fitted with fecal collections bags during the collection period. Feces were collected over a 24 h period, weighed, thoroughly mixed, and subsampled daily. Approximately 250 g of fresh feces was subsampled from the total daily fecal output, partially dried in a forced-air ventilation oven at (60 °C) for 72 h, ground at 1 mm, and pooled by animal and period for subsequent chemical analysis.

2.3. Marker Infusion and Intestinal Digesta Sampling

Chromic oxide was used as an indigestible flow marker to estimate digesta flow. Each steer received a daily dose of 16 g of chromic oxide contained in a gelatin capsule. The capsules were administered through the ruminal cannula at 0700 h each day, starting 5 days prior to intestinal digesta sampling and continuing throughout the collection period (days 13 to 20 of each experimental period).

To represent a complete 24 h cycle, a total of 8 duodenal digesta collections were performed for 3 days (d 18 through d 20 of each experimental period) in 9 h intervals. The digesta collections were conducted as follows: at 0800 h and 1700 h on d 1; at 0200 h, 1100 h, and 2000 h on d 2; and at 0500 h, 1400 h, and 2300 h on d 3. To perform the duodenal samplings, 15.2 × 22.9 cm plastic sampling bags with wire closures were placed at the end of the double L-shaped cannulas, allowing the digesta to flow into the bags. Following collection, the duodenal samples were immediately stored at −20 °C until they were lyophilized, composited per animal per period and ground to 1 mm. The samples were then stored for future chemical analysis.

2.4. Rumen Fluid Collection and Processing

On d 21, rumen digesta was collected at multiple time points to determine fermentation parameters (volatile fatty acids [VFA], ammonia, and lactate). Background samples were obtained at 0500 h (2 h before feeding), with subsequent collections at feeding (0 h) and at 2, 4, 6, 8, 10, and 12 h post-feeding. For each time point, a handful of digesta was obtained from each of the five ruminal sacs (cranial, dorsal, caudodorsal blind, caudoventral blind, and ventral sacs). Digesta was filtered through two layers of cheesecloth to obtain rumen fluid; three 10 mL aliquots were immediately collected and promptly stored (within 1 min of collection) at −20 °C for further analysis.

2.5. Ruminal Bacterial Reference Sampling

On day 28 of each experimental period, total ruminal evacuations were conducted at 1400 h. Ruminal contents were completely removed, weighed, and thoroughly mixed. A representative 4 kg sample of the mixed contents was collected and preserved in 2 L of formalin-saline solution (3.7% formaldehyde and 0.9% NaCl) and stored at −20 °C. This sample served as the reference for the isolation of bacterial cells and subsequent purine analysis to quantify duodenal microbial flow. All contents were returned to the animal immediately following sampling.

2.6. Laboratory Analysis

Feed ingredients, orts, feces, and duodenal samples were analyzed for DM [28], organic matter (OM) [28], crude protein (CP, LECO Corporation, St. Joseph, MI, USA), neutral detergent fiber (NDF, Ankom Technology, Macedon, NY, USA, 1998), and starch [29]. Additionally, feces and duodenal samples were analyzed for chromium oxide content [30].

Ruminal samples were assayed for VFA, ammonia, and lactate. Briefly, acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were determined using a gas chromatograph with FID (Agilent Technologies, Inc., Santa Clara, CA, USA) [31]. Ammonia concentrations were analyzed using a glutamate dehydrogenase procedure [32] to a Konelab 20XTi clinical analyzer (Thermo Fisher Scientific Inc., Beverly, MA, USA). Ruminal L(+)-lactate concentration was measured using the L(+)-lactate dehydrogenase procedure [33,34] adapted to a multi-mode plate reader (BioTek Synergy HTX; Agilent Technologies Inc., Santa Clara, CA, USA).

Bacterial isolation was accomplished using the 4 kg samples collected during rumen evacuation. Samples were centrifuged in 250 mL bottles at 500× g for 20 min to remove protozoa and feed particles. The supernatant was removed and centrifuged at 30,000× g for an additional 20 min to pellet bacteria, which was frozen and lyophilized before being analyzed. Purine concentrations were measured in the duodenal digesta and bacterial samples using the UV/VIS spectrophotometry method to determine the duodenal bacterial flow [35].

2.7. Calculations

Calculations followed previously reported procedures [36]. Concentrations of Cr in the duodenal digesta and feces were used to estimate the DM flow. Duodenal flow (g/d) and fecal excretion (g/d) of nutrients were calculated by multiplying their respective concentrations in the digesta or fecal samples (g/kg DM) by the corresponding daily DM flow at the duodenum or feces. Ruminal digestibility expressed as a percentage of intake was calculated as the difference between intake and duodenal flow, divided by intake. Post-ruminal digestibility, expressed as a percentage of entering the small intestine, was calculated as the difference between duodenal and fecal flows, divided by duodenal flow. Finally, total tract digestibility, expressed as a percentage of intake, was determined as the difference between intake and excretion in the feces, divided by intake.

To calculate duodenal flow of bacterial OM and N, purines were used as a bacterial marker. Duodenal purine flow was calculated by multiplying the daily grams of digesta flow by the percentage of purine RNA in duodenal digesta samples. Duodenal flow of bacterial DM was calculated by multiplying the duodenal purine flow by the percentage of purine RNA present in the ruminal isolate bacteria samples. Finally, the analyzed OM and N concentration of the bacterial pellets were multiplied by the duodenal flow of bacterial DM to calculate the duodenal flow of bacterial OM and N.

2.8. Statistical Analysis

The statistical analysis was conducted using the Mixed Procedure of SAS (version 9.4, SAS Inc., Cary, NC, USA). Animal within period was considered the experimental unit. Degrees of freedom were computed using the Kenward-Rogers approximation. For nutrient intake and digestibility variables, the model included the fixed effects of monensin, essential oils and their interaction, and the random effects of animal and period. The duodenal flow data of two animals were excluded from statistical analysis in periods 3 and 4, due to issues during collection and reduced duodenal flow towards the end of the trial. This did not interfere with the total tract digestibility calculations, but we would like to highlight that for the post-ruminal digestibility calculations, we worked with n = 12 instead of n = 16, which was still enough to detect any potential significant differences. In the case of ruminal fermentation variables, data were analyzed as repeated measures using the Mixed Procedure of SAS. The model included the fixed effects of monensin, essential oils, and their interaction, the random effects of animal and period, and hour for the repeated statement with animal within period as the subject. Covariance structure was selected based on the smallest AICC, with AR(1) and ARH(1) being the chosen ones. When the interaction between monensin and essential oils was significant, mean separation was conducted using Tukey adjustment. Significance was determined at p ≤ 0.05, and tendency was considered when 0.10 > p > 0.05.

3. Results

3.1. Nutrient Intake, Flow, and Digestibility

Data regarding nutrient intake, flow, and digestibility are presented in

Table 2. Effects of an essential oil blend (eugenol, linalool, anethole, and cinnamaldehyde), monensin, or their combination on nutrient intake, flow, and digestibility of Red Angus steers fed a high-grain corn-based finishing diet ¹.

	Treatments 2
	MON−		MON+			p-Value 4
Item	EO−	EO+	EO−	EO+	SEM 3	MON	EO	MON×EO
Dry matter intake, g/d	10.4	10.6	9.76	10.05	0.587	0.08	0.41	0.91
Organic matter
Intake, kg/d	10.01	10.26	9.43	9.67	0.557	0.08	0.39	0.98
Duodenal flow, kg/d	4.94	4.55	4.03	5.48	0.758	0.99	0.40	0.41
Bacterial, g/d	384	338	373	462	54.5	0.12	0.25	0.13
Fecal excretion, kg/d	2.62	2.66	2.26	2.70	0.309	0.40	0.23	0.29
Digestibility
Ruminal, % intake	47.7	51.9	50.4	36.8	4.32	0.18	0.21	0.08
Post-ruminal, % SI 5	55.4	44.5	43.8	47.8	9.07	0.56	0.56	0.39
Total tract, % of intake	74.2	74.4	76.0	72.1	2.32	0.87	0.26	0.23
Starch
Intake, kg/d	4.72	4.50	4.89	4.63	0.502	0.67	0.49	0.96
Duodenal flow, kg/d	1.39	0.90	1.12	0.95	0.324	0.74	0.35	0.64
Fecal excretion, kg/d	0.67	0.42	0.66	0.49	94.70	0.76	0.09	0.70
Digestibility
Ruminal, % intake	72.6	78.4	75.8	81.6	6.77	0.66	0.42	0.99
Post-ruminal, % SI 5	50.7	46.7	36.6	49.9	15.10	0.73	0.77	0.59
Total tract, % of intake	85.9	90.8	86.0	88.8	2.03	0.59	0.05	0.52
Neutral Detergent Fiber
Intake, kg/d	1.94	2.03	1.79	1.92	0.298	0.08	0.13	0.77
Duodenal flow, kg/d	1.31	1.22	1.26	1.5	0.299	0.71	0.80	0.62
Fecal excretion, kg/d	1.02	1.10	0.89	1.03	0.088	0.25	0.19	0.70
Digestibility
Ruminal, % intake	34.5	24.7	21.3	24.0	26.60	0.80	0.90	0.82
Post-ruminal, % SI 5	29.5	13.7	20.0	26.5	13.35	0.91	0.74	0.43
Total tract, % of intake	46.3	42.8	47.3	39.8	9.42	0.78	0.16	0.58
Nitrogen
Intake, g/d	224	227	204	220	22.0	0.07	0.20	0.33
Duodenal flow, g/d	249	221	222	237	32.3	0.83	0.76	0.47
Bacterial, g/d	37	35	30	40	6.0	0.94	0.51	0.51
Fecal excretion, g/d	68	68	60	72	6.2	0.65	0.24	0.27
Digestibility
Ruminal, % intake	−16.0	3.4	−11.6	−19.9	19.89	0.44	0.58	0.36
Post-ruminal, % SI 5	76.0	68.1	72.5	65.5	6.51	0.57	0.20	0.93
Total tract, % of intake	69.6	68.7	70.0	66.7	3.68	0.77	0.46	0.66

¹ Basal diet comprised (all on DM basis): dry-rolled corn 67%, grass hay 10%, dry distillers’ grains 20%, vitamin and mineral premix 3%. ² MON-: no monensin fed; MON+: monensin fed at 400 mg/d; EO-: no essential oils fed; EO+: essential oils blend fed at 14 g/d. ³ Standard error of the mean (n = 4/treatment). ⁴ Observed significances for the effects of: MON (monensin), EO (essential oils), MON×EO (interaction monensin by essential oils). ⁵ Percentage of nutrients entering small intestine.

. No interactions were observed between MON and EO for intake, duodenal flow, or digestibility for any of the nutrients evaluated (p > 0.10), with the exception of a tendency for an interaction observed for OM digestibility (p = 0.08). Supplementation with MON tended to decrease (p ≤ 0.08) the intake of DM, OM, NDF and N, yet starch intake was unaffected by MON supplementation (p = 0.67). No MON effect was observed for duodenal flow or digestibility of any of the nutrients evaluated (p ≥ 0.12). Supplementation with EO tended to decrease (p = 0.09) the fecal excretion of starch by 31.6% and increased (p = 0.05) the apparent total tract digestibility of starch by 3.85 percentage points, or 4.5%. No other effects of EO supplementation were observed for the rest of the nutrients evaluated (p ≥ 0.12).

3.2. Ruminal Fermentation Parameters

Data regarding ruminal fermentation parameters are presented in

Table 3. Effects of an essential oil blend (eugenol, linalool, anethole, and cinnamaldehyde), monensin, or the combination on the ruminal fermentation parameters of Red Angus steers fed a high-grain corn-based finishing diet ¹.

	Treatments 2					p-Value 4
	MON−		MON+							Time×
Item	EO−	EO+	EO−	EO+	SEM 3	MON	EO	MON×EO	Time	MON	EO	MON×EO
(%)
Acetate	56.9	51.8	53.2	50.3	3.46	0.15	0.04	0.50	<0.01	0.87	0.28	0.44
Propionate	22.1	28.0	24.8	33.4	5.76	0.17	0.03	0.62	0.15	0.18	0.29	0.86
Butyrate	12.8	12.8	14.4	11.8	1.74	0.83	0.37	0.35	<0.01	0.52	0.09	0.80
Isobutyrate	1.00	0.88	0.92	0.75	0.113	0.14	0.06	0.68	<0.01	0.79	0.57	0.41
Valerate	2.05	1.54	1.40	1.58	0.202	<0.01	0.08	<0.01	<0.01	0.36	0.30	0.44
Isovalerate	5.18	5.01	5.29	2.16	1.61	0.21	0.14	0.18	0.85	0.15	0.18	0.99
Lactate	0.52	0.99	1.29	0.43	0.361	0.68	0.47	0.02	0.06	0.17	0.23	0.40
Ammonia	9.44	8.48	9.14	8.98	2.61	0.94	0.70	0.78	0.03	0.59	0.59	0.51
A:P 5 ratio	2.57	1.70	1.99	1.45	0.529	0.17	0.03	0.94	0.05	0.22	0.23	0.91

¹ Basal diet comprised (all on DM basis): dry-rolled corn 67%, grass hay 10%, dry distillers’ grains 20%, vitamin and mineral premix 3%. ² MON-: no monensin fed; MON+: monensin fed at 400 mg/d; EO-: no essential oils fed; EO+: essential oils blend fed at14 g/d. ³ Standard error of the mean (n = 4/treatment). ⁴ Observed significances for the effects of MON (monensin), EO (essential oils), MON×EO (interaction monensin by essential oils), Time, Time×MON (time by monensin), Time×EO (time by essential oils), Time×MON×EO (triple interaction). ⁵ Acetate to propionate ratio.

. No interactions between time with MON, time with EO, and time with MON and EO were observed for any fermentation parameters evaluated (p ≥ 0.09). The time of collection affected the percentage of acetate, butyrate, isobutyrate, valerate, and ammonia, and the acetate to propionate ratio (p ≤ 0.05), and tended to affect lactate (p = 0.06). Propionate concentration was not affected by time of collection (p = 0.15). An interaction between MON and EO was observed for valerate concentrations (p < 0.01), where the MON-EO- had the greatest concentration, followed by the rest of the treatments, and these last three did not differ among them (p ≥ 0.46). Therefore, the main effect of MON (p < 0.01) and a tendency of EO (p = 0.08) for valerate concentrations are disregarded, and differences for this parameter are considered as interactions between the main factors. Also, an interaction between MON and EO was observed for lactate concentrations (p = 0.02); yet, when means were separated and differences adjusted by Tukey, no differences among treatments were observed (p ≥ 0.13).

No other effects of MON were observed on the ruminal fermentation parameters evaluated (p ≥ 0.15). Supplementation with EO decreased acetate concentrations by 7.3% (p = 0.04) and tended to decrease isobutyrate concentrations by 15.1% (p = 0.06). Also, supplementation with EO increased propionate concentrations by 30.9% (p = 0.03). Thus, the A:P ratio decreased by 30.9% (p = 0.03) with EO supplementation. No other effects of EO were observed on the rest of the ruminal fermentation parameters evaluated (p ≥ 0.14).

4. Discussion

To the best of our knowledge, this is the first article published evaluating the use of a blend of eugenol, linalool, anethole, and cinnamaldehyde in feedlot diets. While there is a lack of previous studies published with this specific blend, there is significant information available regarding the use of EOs in ruminant diets. However, it is important to note that the efficacy of these compounds can be highly variable, as it is influenced by factors such as chemical composition, extraction methods, dosage, and diet composition, as well as ruminal conditions and microbial adaptation. It is noteworthy that no relevant interactions were observed between monensin and the botanical supplement evaluated herein. The absence of an interaction effect is relevant because it indicates that the botanical blend can be combined with monensin in feedlot diets without negatively affecting nutrient intake and digestibility.

As expected, supplementation with monensin sodium resulted in a slight reduction in feed intake, as several previous studies have reported [6,17]. Interestingly, we failed to detect differences in nutrient digestibility and ruminal fermentation with the supplementation of monensin, which contrasts with the findings from previous studies [37,38,39]. Yet, trends for greater propionate concentration suggest that a larger number of replicates would be needed to assess the effects of this ionophore in our experimental conditions.

The 4.5% greater apparent total tract digestibility of starch with the EOs supplementation is probably the most remarkable finding of this study. The primary starch source in the basal diet was dry-rolled corn grain (67% inclusion in the diet), which is well known to be less degradable than high-moisture corn or steam flaked corn [40]. While we failed to detect an improvement in ruminal starch degradation with EO supplementation, the observed means show a trend in that direction, with averages of 74 vs. 80% ruminal starch degradation for the EO− vs. EO+ groups. Moreover, given the increased total tract starch digestibility observed with EOs, and considering the complexity of the determination of ruminal degradation of starch, which involves complex samplings and markers of digesta flow, an improvement in the ruminal degradation of starch should not be ruled out with this study only.

The impact of the EOs on starch utilization could be related to reductions in the protozoa counts caused by the blend of EOs, as demonstrated in two previous meta-analyses with in vivo data [15,41]. It is well known that protozoa capture starch granules and decrease their rate of degradation in the rumen, reducing the rate of degradation of starch in the rumen [42,43]. Yet, in the current study, protozoa populations were not evaluated; therefore, this hypothesis cannot be proved. Another consideration is that dietary supplementation with EOs can reduce the amount of Gram-positive bacteria in the rumen [11]. A reduction in the Gram-positive bacteria population could have facilitated the growth of Gram negatives, and in the rumen, three out of the four major starch-degrading bacteria (Ruminobacter amylophilus, Succinimonas amylolytica, and Selenomonas ruminantium) are Gram-negative [2,44]. While the ruminal microbial population was not directly quantified in the present study, an evaluation of the effects of the EO blend on the ruminal microbiome is currently being conducted by our research group and could help explain some of the findings of this study.

The lack of an effect of the EO blend on the digestibility of OM, NDF, and N is aligned with previous in vivo and in vitro data [41,45]. However, it is important to consider that the antimicrobial effects of EOs vary meaningfully depending on the main compound utilized; they are dose-dependent, and can have differential effects depending on the basal diet fed [11]. Therefore, these findings should be restricted to this specific product evaluated, at this specific dose, and under a high-grain diet.

Feeding the EO blend for as little as 14 days was enough to shift fermentation patterns towards an increased concentration of propionate. The observed changes on ruminal fermentation patterns, with reduction in the A:P ratio, are consistent with the known ruminal effects of eugenol [18,21], cinnamaldehyde [11,25], and anethole [11,22,23]. The greater propionate and reduced A:P ratio in this trial by feeding the EO blend aligns with the findings from a meta-analysis conducted by researchers at the University of Vienna [15]. Their results showed that supplementation with blends of EO to beef cattle reduced the molar proportion of acetate by up to 10%, and increased the molar proportion of propionate, with a subsequent decrease in the acetate to propionate ratio [15]. Furthermore, findings from that meta-analysis suggest improved utilization of dietary starch when feeding EOs, since dairy cows fed essential oils exhibited improved milk protein concentration and milk protein yield [15]. Similarly, a more recent meta-analysis conducted in 2022 by researchers from Mexico assessing data from beef cattle studies supplemented with EOs [41] agrees with our findings. In their meta-analysis, the weighed means from 14 trials demonstrated that supplementation with EOs reduces acetate on average by 7.1% and increases propionate on average by 3.7% [41]. Furthermore, a meta-analysis from in vitro assays demonstrated a reduction in acetate concentration with EO addition in the substrate [45].

The lack of an effect on other fermentation variables from supplemental EOs in this experiment is opposed to previous meta-analysis from in vivo trials, where feeding EOs decreased ruminal ammonia nitrogen [41] and decreased ruminal butyrate concentration [15], in addition to results from a meta-analysis of in vitro trials where EOs decreased ruminal ammonia concentration [45]. The lack of an effect on ammonia nitrogen could be related to the N sources in the basal diet. While urea supplementation can influence the ruminal microbial population and diversity, it is completely solubilized in the rumen and is not expected to be impacted by a feed additive that targets proteolytic pathways. Also, protein from DDGS has a low ruminal degradability [~32% ruminal degradable protein (RDP)], as compared to other sources like soybean meal (~70% RDP) [1]. Additionally, the low ruminal pH commonly observed under finishing diets has a negative impact on bacterial proteases activity, resulting in reduced protein degradation [46]. Therefore, a diet containing true plant protein with greater RDP may be a better experimental model to evaluate the effects of the EO product on ruminal protein degradation. While urea is a standard and practical N source in feedlot formulations, its rapid hydrolysis may mask the specific protein-sparing effects of EOs, which are more readily observed in diets where a higher proportion of ruminal ammonia is derived from the degradation of true protein.

Altogether, the improved starch utilization and increased propionate production experienced when feeding the blend of eugenol, linalool, anethole, and cinnamaldehyde clearly suggest that this blend of EOs could enhance growth performance at the feedlot. Previous meta-analysis data also indicates that feeding blends of EOs in finishing diets can increase final body weight average daily gain, feed conversion rate, hot carcass weight, and ribeye area [41]. Therefore, the results from our trial, supported by previous studies, make the botanical blend evaluated herein a solid candidate for performance trials, and suggest it could be of great value for feedlot diets based on dry-rolled corn.

5. Conclusions

Results from this experiment do not suggest a negative effect of combining monensin sodium with the evaluated blend of eugenol, linalool, anethole, and cinnamaldehyde. Feeding monensin sodium tended to decrease feed intake, as expected. Feeding the blend of EOs improved the utilization of starch in a corn-based high-grain finishing diet, increased the ruminal concentration of propionate, and decreased the concentration of acetate and the ratio of acetate to propionate. Altogether, it appears that this EO blend could be helpful for feedlot diets to increase the utilization of starch, particularly when high-moisture corn and steam-flake corn are not feasible.