A Comprehensive Analysis of Cinnamon, Flaxseed, and Lemon Seed Essential Oils’ Effects on In Vitro Gas Formation and Nutrient Degradability in Diets

Abstract

This study aimed to assess the impact of essential oils (EOs) on in vitro gas formation and the degradability of dairy and beef cattle diets. This study also aimed to investigate the effects of different types of EOs on nutrient utilization and rumen microbial activity. The current study was conducted using a fully randomized design consisting of eight experimental treatments, including two control treatments without any additives, and treatments with cinnamon essential oil (CEO), flaxseed essential oil (FEO), and lemon seed essential oil (LEO) at a concentration of 60 mg/kg fresh mass. Two control treatments were used, one with alfalfa silage and dairy concentrate (DC, CON-DC) and the other with alfalfa silage and fattening concentrate (FC, CON-FC). Gas formation, dry matter (DM) digestibility, crude protein (CP) digestibility, effective degradability (ED), and soluble fractions of DM and organic matter (OM) were evaluated. CEO had a substantial effect on gas formation (p < 0.05). When EOs were added to the diets, they increased dry matter digestibility after 24 h of incubation as compared to control treatments. After 24 h of incubation, FCCEO and FCFEO had the highest CP digestibility among the diets. FCLEO considerably enhanced ED, as well as the soluble fraction of DM (a) at a passage rate of 2% per hour. Treatment with FCCEO resulted in a significant increase in soluble fractions compared to the control diets. At a passage rate of 2% h, DCCEO had the maximum ED value. When EOs were introduced to the diet, they dramatically decreased the insoluble portion of CP (b). Compared to the control treatments, gas production was significantly lower in the presence of LEO (FCLEO; p < 0.05). The addition of EOs to cattle diets may increase nutrient utilization and enhance rumen microbial activity. EOs extracted from lemon seeds (at a dose of 60 mg/kg of diet) lowered gas production in both dairy cattle and fattening diets.

1. Introduction

Increasing attention has been paid to medicinal plants due to advances in organic chemistry. This is due to changes in the methods of extracting, purifying, and analyzing plants’ effective compounds, as well as the side effects of chemical drugs [1]. Now, approximately one third to one half of drug products are made from medicinal plants [1]. In many countries, plants are used to make drugs. Moreover, the excessive use of chemical drugs and antibiotics in animals has threatened human health by their presence, and that of their degradation products, in water and food, as well as by the development of antibiotic resistance (i.e., the formation of multi-resistant germs) [1]. Hence, more and more bans have been enacted against antibiotics in livestock and poultry feeds, particularly when used as growth promoters and for prophylactic use [1].

Generally, essential oils (EOs) are volatile and aromatic compounds that are derived from plants [2]. EOs are primarily composed of terpenoids and phenylpropanoids, both active compounds. These compounds exhibit a wide range of structures, natures, and functions. Due to this, they have long been used as food preservatives as well as in traditional medicines [3]. A major role for these compounds is their antiseptic and antimicrobial [4] properties, which are mediated by the hydrophobic nature of their hydrocarbon rings, which interfere with the membranes of bacteria. In bacteria, these compounds accumulate between fatty acid chains and occupy the spaces between them [5]. This interferes with the cell membrane, resulting in increased fluidity [6]. During membrane instability, ions leak out of the cell wall and reduce the ion transfer gradient, so the bacterium maintains an ionic balance by using an ion pump. In this condition, the bacterial cell does not die, but grows slowly as most of its energy is used to maintain an ionic balance [7]. In addition, previous studies have demonstrated that various EOs and their active compounds could positively influence rumen microbial metabolism [8,9]. As evidenced by recent studies [10], a combination of EOs in the form of CRINA^® Ruminants, when paired with amylase enzymes, can offer a viable alternative to ionophore antibiotics in promoting the production and quality of milk in dairy cows. Furthermore, the replacement of antibiotics with a thymol, eugenol, and vanillin blend of EOs in the finishing process of cattle has demonstrated comparable results [11].

Biological activity has been found in EOs, secondary metabolites that produce plant odor, color, and taste [12]. EOs, including their active compounds, are believed to have antimicrobial properties due to the presence of phenolic compounds and terpenoids [13]. This characteristic impacts a wide range of microorganisms, encompassing Gram-negative bacteria, Gram-positive bacteria, yeasts, and fungi [14]. Therefore, EOs may alter and modify the fermentation process in silage, ultimately improving the fermentation pattern and reducing ruminant emissions in silage by altering the microbial population [15]. According to Naseri et al. [16], supplementation with the Pistacia atlantica subspecies Kurdica gum essential oil increased the total ruminal bacterial count in ruminants. By contrast, total rumen methanogens declined, and protozoa did not change. Molero et al. [17] conducted a study investigating essential oils (EOs) utilization in beef cattle feed. Similarly, Besharati et al. [15] compared the quality and fermentation characteristics of Lucerne silage supplemented with flaxseed, cinnamon, and lemon seed EOs. They reported that adding EOs improved silage quality and fermentation characteristics. This study expands upon prior research and evaluates the effects of cinnamon essential oil (CEO), lemon seed essential oil (LEO), and flaxseed essential oil (FEO) on in vitro gas formation, as well as the degradability of dry matter (DM), organic matter (OM), crude protein (CP), and neutral detergent fiber (NDF) in diets for both dairy and beef cattle.

2. Materials and Methods

This study was conducted at the University of Tabriz, Iran. In vitro measurements were conducted on gas formation and digestibility during the experiment.

2.1. Silage Preparation

In this study, fourth-cut alfalfa (lucerne, Medicago sativa L.) samples were collected in a field in Tabriz province, Iran, in late summer 2022, chopped into 3–5 cm pieces, and ensiled for 60 days in lab-scale tubes (3.00 ± 0.25 kg) after 24–30 h wilting [18].

2.2. Essential Oils Preparation

This study examined cinnamon, flaxseed, and lemon seeds. The raw materials for this study were procured from local markets in Ahar and Tabriz and subjected to a standardized preparation process. Plant samples were cut or crushed into small pieces and dried at 39 °C for 48 h before being ground to a particle size that passed through a 1 mm screening screen. The essential oils were extracted from the ground plant samples using a Clevenger apparatus [19] and subsequently stored in a refrigerator at 4 °C until they were required for the experiment.

2.3. Experimental Diets

The experimental diets, including a dairy cattle diet and a beef cattle diet, were prepared with alfalfa silage: concentrate ratios of 40 to 60 or 20 to 80, respectively, as recommended by the National Research Institute [20]. Based on the chemical composition of the basic diets shown in Table 1, the dried experimental diets were milled through a sieve mill with a 1-mm mesh size [21]. The eight treatment groups used in this study included CON-DC, a control group consisting of alfalfa silage (40%) and dairy concentrate (60%) without any additives; CON-FC, another control group consisting of alfalfa silage (20%) and fattening concentrate (80%) without any additives; DCCEO, the first experimental group, consisting of CON-DC with the addition of 60 mg/kg fresh mass of cinnamon essential oil; FCCEO, the second experimental group, consisting of CON-FC with the addition of 60 mg/kg fresh mass of cinnamon essential oil; DCFEO, the third experimental group, consisting of CON-DC with the addition of 60 mg/kg fresh mass of flaxseed essential oil; FCFEO, the fourth experimental group, consisting of CON-FC with the addition of 60 mg/kg fresh mass of flaxseed essential oil; DCLEO, the fifth experimental group, consisting of CON-DC with the addition of 60 mg/kg fresh mass of lemon seed essential oil; and FCLEO, the sixth experimental group, consisting of CON-FC with the addition of 60 mg/kg fresh mass of lemon seed essential oil. The essential oils were added directly to bottles using a micropipette and mixed.

Table 1. Ingredients of the 2 concentrates and chemical compositions of diets’ ingredients which were incubated in vitro (% of dry matter).

Ingredients	Dairy Concentrate (DC)	Fattening Concentrate (FC)	Alfalfa Silage (AS)
Wheat bran	15	18	-
Corn grain, ground	11	10	-
Milo **	10	4	-
Soybean meal	2	3	-
Barley grain, ground	15	10	-
Meat powder (60% CP)	5	6	-
Urea	0.6	1	-
Calcium carbonate	2.5	2.5	-
Molasses	3	3	-
Rice bran	27	27	-
Salt (NaCl)	0.5	0.5	-
Di-calcium phosphate	0.4	0	-
Mineral-vitamin mix ***	2	2	-
Fruit pulp (apple and orange)	4	11	-
Calcinit *	2	2	-
Chemical composition
Dry matter (%)	92.60 ± 0.44	91.49 ± 0.34	24.40 ± 0.63
Crude protein (% DM)	22.40 ± 0.70	20.02 ± 0.70	11.62 ± 0.43
Crude ash (% DM)	16.66 ± 0.34	20.22 ± 0.70	11.40 ± 0.03
Acid detergent fiber (% DM)	6.40 ± 0.15	5.20 ± 0.02	22.60 ± 1.40
Neutral detergent fiber (% DM)	20.40 ± 1.02	18.60 ± 0.34	49.07 ± 1.78
pH	-	-	4.35 ± 11

* Nitrate source with molecular formula Ca(NO₃)₂. ** Grain sorghum (Sorghum bicolor (L.) Moench). *** Each kg of mineral-vitamin mix contained 19 g of calcium, 90 g of phosphorus, 50 g of sodium, 19 g of magnesium, 3 g of iron, 300 mg of copper, 2000 mg of manganese, 3000 mg of zinc, 100 mg of cobalt, 100 mg of iodine, 300 mg of antioxidants, 1 mg of selenium, 500,000 international units (IU) of vitamin A, 100 mg of vitamin E, and 100,000 IU of vitamin D3.

Chemical Analysis

The chemical compositions of the alfalfa silage, dairy concentrate, and fattening concentrate, as well as the pH levels of the samples, were evaluated in accordance with established protocols [22]. In order to measure dry matter (DM), the samples were dried in an oven for 48 h at 65 °C, followed by grinding using a sieve with a diameter of 1 mm. The DM, crude ash (CA), and crude protein (CP) contents were determined using the methods outlined in AOAC [22]. The determination of CP was carried out using method ID 984.13. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured following the guidelines of Van Soest et al. [23], without the use of amylase and sodium sulfite. The NDF, which included ash, was analyzed with/without amylase. To extract aqueous samples of ensiled materials, 20 g of forage were homogenized with 180 mL of water (deionized) for one minute. The pH of the rumen liquid for serum bottles at the end of incubation was measured, and the acidified extracts were treated with Ammonia-N (NH₃-N) using the Kjeldahl method [24].

2.4. In Vitro Gas Formation Test

At a slaughterhouse, three slaughtered sheep were sampled for ruminal fluid [25]. A sealed thermos was used to transport squeezed ruminal fluid to the laboratory. To produce gas in vitro, the method of Fedorah and Hrudy [6] was used. Each serum bottle was filled with 20 mL buffered rumen fluid with McDougal [26] buffer (with a ratio 2:1 of buffer: rumen fluid). Approximately 0.3 g of the samples (dried and ground) were placed in the bottles. There were 5 replications for each treatment. Gas formation was monitored over the incubation period at 2, 4, 6, 8, 12, 16, 24, 36, and 48 h, and recorded accordingly. A triplicate measurement of gas formation profiles was conducted using the equation Y = A(1 − e^−ct), where Y represents the gas produced (in mL) per gram of dry matter (DM) at time t, A represents the gas production from both soluble and insoluble fractions, c represents the gas formation rate, and t represents the incubation time (in hours) [27]. Using the equations established by Menke et al. [28], the metabolizable energy (ME) content of gas formation (GP) and organic matter digestibility (OMD) was determined. Additionally, the following equation was applied to predict short-chain fatty acid (SCFA) formation (see also Besharati and Niazifar (2020)):

SCFA = 0.0222 × GP − 0.00425

(1)

In Equation (1), GP is the gas formation at 24 h (mL/200 mg DM). SCFAs are measured in mmol. For details, see [25].

2.5. In Vitro Digestibility

Rumen fluid collection was performed as described previously [29]. Each serum bottle was filled with 20 mL buffered rumen fluid with McDougal [26] buffer (with a ratio 2:1 of buffer: rumen fluid). Approximately 0.3 g of samples (dried and ground) were placed in the bottles. Data were sampled after 2, 4, 8, 12, and 24 h of incubation. The residue of treatments after selected times was collected and washed 3 times with buffer phosphate and then dried. There were 3 replications for each treatment for each time. For each treatment, the degradation was determined using Naway software and by fitting the measured data to a non-linear function: Y = a + b(1 – e^−ct).

Y represents the volume of degradability at time t, a + b represents the fermentation rate for soluble and insoluble fractions (mL/200 mg DM), and c represents the constant fractional rate of fermentation [29].

PD = a + b

(2)

ED = a + b × c/(c + 0.05)

(3)

The degradability was measured using 24 h fermentation (mL/200 mg DM), and the CP, CF, and CA were expressed in terms of percent DM.

Net energy for lactation (NEL; MJ/kg dry matter) was calculated using the following equation [30]:

NEL (MJ/kg DM) = 0.36 + 0.156 GP + 0.0054 CP + 0.014 EE + 0.0054 ASH

GP: volume of gas produced in 24 h (mL/200 mg DM); EE: Ether extract (%); and ASH: crude ash (%).

2.6. Statistical Analysis

The data collected from the in vitro gas formation and digestibility experiments were analyzed using a completely randomized design and the GLM procedure of SAS [31]. This model was used: Y_ij = μ + T_i + e_ij

Here, Y_ij stands for the dependent variable, and μ is the mean. T_i stands for the impact of treatment (i.e., the addition of essential oils) and e_ij is random error. The gas formation and in vitro digestibility tests were repeated 5 and 3 times, respectively. Statistical evaluation of the data was done with the Duncan test, and differences between treatments were considered significant if the p-value was less than 0.05.

3. Results and Discussion

The alfalfa silage and concentrates used are listed in Table 1. The crude protein, acid detergent fiber, and neutral detergent fiber contents of the dairy concentrate and fattening concentrate were 22.40 vs. 20.02%, 6.40 vs. 5.20%, and 20.40 vs. 18.60%, respectively.

3.1. In Vitro Gas Formation

Table 2 and Table 3 display the influence of the essential oils (EOs) on gas formation and estimated parameters from the experimental diets. The results showed that cinnamon essential oil (CEO) and lemon seed essential oil (LEO) had a statistically significant effect on gas formation (p < 0.05). The treatment FCLEO showed the lowest gas formation compared to the control treatments (CON-DC and CON-FC), indicating a significant impact of EO on gas formation. It is imperative to note that the amount of gas produced is directly related to the digestion of organic matter, which creates a favorable environment for ruminal bacteria to flourish. Diets rich in digestible carbohydrates (e.g., those with higher levels of concentrate) promote greater fermentation of substrates, enabling rumen bacteria to produce significant amounts of volatile fatty acids, compared to those composed of structural carbohydrates [32]. Therefore, the present study suggests that EOs significantly influence in vitro gas formation, confirming the findings of previous in vitro studies. According to Patra et al. [33], the use of EOs in vitro reduces gas formation, while Tawab et al. [34] reported that EOs reduced gas formation over a 24 h incubation period. It is crucial to consider how secondary plant compounds are extracted to determine their effect. There is also the possibility that the reduction of gas formation is due to some EOs of the studied EOs having antimicrobial properties. These properties limit microorganism activity, preventing gas formation [35]. On the one hand, this reduction indicates a decrease in organic matter fermentation, but on the other hand, it can indicate the movement of the material towards microbial production, so it can improve feed efficiency. Methane-producing bacteria have increased in fodder diets, resulting in increased methane production compared to concentrates [35].

Table 2. Essential oils and their effects on gas formation in experimental diets (mL/g DM).

Treatments	Times (h)
	2	4	6	8	12	16	24	36	48	72	96	120
CON-DC	14.81 a	25.94 a	38.96 a	45.64 a	59.84 a	76.07 a	102.67 a	107.38 a	118.09 a	127.49 a	129.39 a	132.06 a
CON-FC	14.34 a	25.64 a	38.66 a	44.73 a	59.71 a	75.98 a	98.97 a	106.92 a	117.10 a	126.69 a	129.22 a	131.97 a
DCCEO	11.89 b	20.47 c	27.87 c	39.39 c	54.64 c	71.74 c	94.38 c	103.11 c	110.38 bc	118.70 d	122.23 b	125.13 b
FCCEO	12.62 b	23 b	38.19 b	43.72 b	59.11 b	75.34 b	93.92 c	104.65 c	113.31 b	120.77 c	124.50 b	126.59 b
DCFEO	14.72 a	23.56 b	28.96 c	40.82 bc	55.34 c	73.84 c	98.80 b	106.19 b	116.32 b	125.11 b	129.04 a	131.52 a
FCFEO	14.25 a	23.03 b	29.70 bc	43.49 b	57.41 bc	72.71 c	86.89 d	97.69 d	106.82 c	113.54 e	116.80 c	123.83 c
DCLEO	12.45 b	21.17 c	27.84 c	39.69 c	53.48 d	69.39 d	86.29 d	95.89 d	103.69 c	110.33 e	110.51 d	115.89 d
FCLEO	10.65 c	19.78 d	23.64 d	34.97	48.75 e	63.86 e	71.30 e	78.90 e	87.84 d	64.63 f	98.42 e	118.19 d
SEM	0.9342	2.576	2.695	2.353	2.048	2.065	2.365	2.707	2.960	3.107	2.848	2.320
p-value	0.0003	0.003	0.0018	0.0003	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001

CON-DC: Control 1, alfalfa silage (40%) + dairy concentrate (60%) without additives; CON-FC: Control 2, alfalfa silage (20%) + fattening concentrate (80%) without additive; DCCEO: CON-DC + 60 mg/kg fresh mass of cinnamon essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of cinnamon essential oil; DCFEO: CON-DC + 60 mg/kg fresh mass of flaxseed essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of flaxseed essential oil, DCLEO: CON-DC + 60 mg/kg fresh mass of lemon seed essential oil; FCLEO: CON-FC + 60 mg/kg fresh mass of lemon seed essential oil. Different superscripts within the same column indicate different means (p < 0.05).

Table 3. Impact of essential oils on estimated parameters obtained from gas formation of treatments.

Treatments 1	Parameters
	pH	SCFA	ME	NEL	OMD	DOMD	NH3-N
CON-DC	6.63 b	0.101 d	3.360 a	1.315 a	25.073 d	25.043 a	50.458 c
CON-FC	6.70 a	0.095 d	3.293 a	1.297 a	26.047 d	24.413 a	46.912 d
DCCEO	6.50 c	0.152 ab	3.224 ab	1.259 b	29.743 a	24.011 ab	50.458 c
FCCEO	6.55 bc	0.140 b	3.16 b	1.216 b	28.538 b	23.318 b	60.314 a
DCFEO	6.47 c	0.158 ab	3.282 ab	1.297 b	29.293 ab	23.936 ab	47.340 d
FCFEO	6.57 bc	0.1018 c	3.942 c	0.972 c	27.453 c	23.163 b	42.5 d
DCLEO	6.71 b	0.155 ab	2.66 d	0.876 d	25.583 d	21.585 c	47.184 d
FCLEO	6.68 b	0.169 a	2.668 d	0.853 d	27.229 c	21.631 c	52.222 b
SEM	0.055	0.009	0.045	0.0362	0.164	0.3410	0.2631
p-value	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001	≤0.0001

SCFA: Short-chain fatty acid (mmol/200 mg DM); ME: metabolizable energy (MJ/kg DM); NEL: net energy lactation (MJ/kg DM); OMD: organic matter digestibility (%); DOMD: digestible organic matter in dry matter (%); NH₃-N: ammonia nitrogen (mg/dL); ¹ CON-DC: Control, alfalfa silage (40%) + dairy concentrate (60%) without additives; CON-FC: Control 2, alfalfa silage (20%) + fattening concentrate (80%) without additive; DCCEO: CON-DC + 60 mg/kg fresh mass of cinnamon essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of cinnamon essential oil; DCFEO: CON-DC + 60 mg/kg fresh mass of flaxseed essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of flaxseed essential oil; DCLEO: CON-DC + 60 mg/kg fresh mass of lemon seed essential oil; FCLEO: CON-FC + 60 mg/kg fresh mass of lemon seed essential oil. The pH was measured at the end of incubation. If the column’s means have different superscripts, there is a significant difference (p < 0.05) between them.

The finding implies that the addition of EOs to experimental treatments significantly affected gas formation at all time points (p < 0.05), whereas gas formation in the CON treatments remained unaffected (p < 0.05). In vitro gas formation comprises carbon dioxide and methane gases, which are produced through microbial fermentation, or when volatile fatty acids react with bicarbonate [36]. As such, gas formation is directly proportional to the amount of digested mass [37]. Moreover, gas formation has a linear correlation with both microbial synthesis and volatile fatty acid net production [12]. According to Foggi et al. [38], adding 67 mg/L of each EO source (10 g/kg) resulted in reduced total volatile fatty acid production and a slight negative impact on IVOMD. However, no mitigation effects on CH₄ or NH₃ formation were observed. The GP, ME, SCFA, DOMD, and OM were affected by EOs. EOs are also reported to maintain the level of nutrients in the silage. Bayatkouhsar et al. [5] found that EOs could affect the amount of gas produced.

3.2. In Vitro Digestibility

The results of digestibility values and parameters are shown in Table 4 and Table 5. After adding LEO, FEO, or CEO, one can see the results of digestibility values and parameters for all the treatments. All the treatments showed higher digestibility rates for OM (organic matter) and DM (dry matter). The experiment with lemon seed essential oil (LEO) exhibited an increase in OM fermentation after 24 h of incubation, while the control treatment showed a decrease in fermentation (p < 0.05). The results also revealed that, after 2 h of incubation, the control treatments had the highest CP digestibility rate, whereas the LEO experiment had the lowest value (p < 0.05). However, LEO was associated with the highest fermentation rate 24 h after incubation, which was significantly different from other treatments.

Table 4. Impact of essential oils on in vitro nutrient digestibility of treatments (%).

Treatments 1	Time of Incubation (h)
	2	4	8	12	24
DM
CON-DC	32.87 d	46.10 e	57.62 c	66.64 c	73.64 c
CON-FC	34.28 d	53.59 e	63.73 c	68.73 c	75.27 c
DCCEO	38.64 b	50.37 b	62.34 b	71.80 a	82.19 ab
FCCEO	39.51 b	53.94 b	64.02 b	74.80 a	83.15 a
DCFEO	43.15 a	45.21 c	58.24 d	67.97 b	75.82 b
FCFEO	44.32 a	59.64 a	66.31 a	74.25 a	83.63 a
DCLEO	36.56 bc	48.14 bc	61.45 c	69.53 b	77.48 b
FCLEO	37.77 a	56.84 a	64.57 b	72.85 ab	84.45 b
SEM	0.5354	0.731	0.256	0.646	0.425
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
OM
CON-DC	30.31 d	40.89 a	54.45 a	62.75 a	71.75 b
CON-FC	29.31 b	40.19 a	49.52 c	60.45 d	67.45 c
DCCEO	28.67 c	32.39 c	51.42 b	57.89 b	65.64 c
FCCEO	29.65 b	38.74 b	54.87 a	61.78 ab	78.56 a
DCFEO	30.18 a	36.97 bc	46.84 c	57.46 b	72.45 b
FCFEO	30.87 a	38.60 b	49.16 c	62.45 ab	64.25 c
DCLEO	29.89 b	37.34 bc	50.56 b	62.17 ab	73.92 b
FCLEO	30.62 a	40.94 a	54.72 a	65.93 a	74.80 b
SEM	0.0347	0.0574	0.0692	0.0488	0.0761
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
CP
CON-DC	21.32 c	28.38 a	31.84 b	36.59 c	41.84 e
CON-FC	22.63 b	27.63 b	32.84 c	35.09 c	44.62 d
DCCEO	21.39 c	27.56 b	32.24 c	36.17 c	45.56 b
FCCEO	22.74 b	28.17 a	34.87 a	36.87 c	47.64 a
DCFEO	22.97 b	26.41 c	32.85 c	36.56 c	42.45 c
FCFEO	23.60 a	27.09 ab	32.43 c	38.41 a	46.16 a
DCLEO	21.34 c	28.43 a	32.12 c	37.53 b	43.58 c
FCLEO	19.06 d	21.49 d	30.45 d	34.24 d	44.81 d
SEM	0.051	0.0765	0.047	0.154	0.0616
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
NDF
CON-DC	22.93 a	22.69 a	32.91 a	37.17 b	47.36 c
CON-FC	23.32 a	25.98 a	32.33 a	37.43 b	49.92 c
DCCEO	19.80 c	21.50 d	25.77 d	32.21 d	48.84 c
FCCEO	21.80 b	24.18 b	31.57 b	35.17 c	50.62 b
DCFEO	19.06 c	22.11 c	29.03 c	32.77 d	45.10 d
FCFEO	21.96 b	26.42 a	32.51 a	37.43 a	52.03 a
DCLEO	18.76 d	22.20 c	26.54 cd	32.60 d	43.17 e
FCLEO	22.93 b	25.58 a	31.80 b	36.50 b	50.93 b
SEM	0.301	0.535	0.0335	0.465	0.0524
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

^a–d There is a significant difference between the averages indicated by different letters of the same column. ¹ CON-DC: Control 1, alfalfa silage (40%) + dairy concentrate (60%) without additives; CON-FC: Control 2, alfalfa silage (20%) + fattening concentrate (80%) without additive; DCCEO: CON-DC + 60 mg/kg fresh mass of cinnamon essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of cinnamon essential oil; DCFEO: CON-DC + 60 mg/kg fresh mass of flaxseed essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of flaxseed essential oil; DCLEO: CON-DC + 60 mg/kg fresh mass of lemon seed essential oil; FCLEO: CON-FC + 60 mg/kg fresh mass of lemon seed essential oil. The notation of different superscripts within the same column indicates a significant difference (p < 0.05) between means. In this table, DM refers to dry matter, OM refers to organic matter, CP refers to crude protein and NDF refers to neutral detergent fiber.

Table 5. Impact of EOs on digestibility parameters of treatments.

Treatments 1	Items
DM	a	b	c	PD	ED
CON-DC	21.24 e	49.34 bc	0.12 c	71.61 d	64.89 d
CON-FC	19.95 e	50.01 bc	0.12 c	72.75 d	71.91 d
DCCEO	18.97 d	60.90 a	0.14 b	79.87 b	70.87 b
FCCEO	19.60 d	60.64 a	0.14 b	80.24 ab	73.24 ab
DCFEO	23.23 c	50.19 bc	0.18 a	73.42 c	66.42 c
FCFEO	25.63 c	54.00 b	0.095 d	79.63 b	72.63 ab
DCLEO	33.97 b	40.51 d	0.17 ab	74.48 c	67.4 b
FCLEO	34.84 a	48.61 c	0.18 a	83.45 a	76.45 a
SEM	1.664	1.051	0.009	1.068	0.851
p-value	0.0003	<0.0001	<0.0001	<0.0001	<0.0001
OM
CON-DC	17.38 c	55.52 a	0.068 e	64.76 d	54.14 d
CON-FC	18.94 c	55.22 a	0.067 d	62.05 d	52.28 d
DCCEO	18.35 b	48.29 c	0.091 c	66.64 c	57.64 c
FCCEO	19.82 a	54.81 b	0.067 d	74.63 a	67.63 a
DCFEO	18.53 b	43.92 d	0.11 b	73.15 a	64.45 b
FCFEO	19.50 ab	42.75 d	0.043 e	62.25 d	55.25 d
DCLEO	19.78 a	51.84 c	0.114 a	71.62 b	64.62 b
FCLEO	18.93 b	53.87 b	0.058 d	72.80 b	65.8 b
SEM	0.0155	0.301	0.008	0.0329	0.342
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
CP
CON-DC	9.92 d	53.17 a	0.09 c	45.64 c	51.13 b
CON-FC	9.83 d	54.94 a	0.09 c	54.63 b	56.12 a
DCCEO	10.87 c	39.24 c	0.07 c	53.56 b	46.56 c
FCCEO	12.39 b	33.25 d	0.15 a	45.64 c	35.64 d
DCFEO	10.8 c	33.65 d	0.11 b	44.45 c	34.45 d
FCFEO	9.21 d	55.95 a	0.10 c	65.16 a	50.16 b
DCLEO	9.80 d	45.78 b	0.08 d	55.58 b	46.58 c
FCLEO	14.32 a	53.93 ab	0.07 c	64.80 a	51.80 b
SEM	0.345	1.491	0.075	0.0814	1.621
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
NDF
CON-DC	28.69 b	54.32 a	0.042 c	65.21 b	45.48 d
CON-FC	29.71 a	44.67 a	0.039 c	63.64 b	44.98 d
DCCEO	27.40 c	52.41 ab	0.051 a	78.84 a	45.95 c
FCCEO	28.52 b	42.10 b	0.046 ab	70.62 ab	47.13 b
DCFEO	26.22 d	28.88 d	0.027 d	55.10 d	45.82 c
FCFEO	28.80 b	33.23 c	0.041 b	62.03 b	50.05 a
DCLEO	29.42 a	23.75 e	0.031 d	53.17 e	45.04 c
FCLEO	27.90 c	33.04 c	0.042 b	60.93 c	47.57 b
SEM	0.435	0.247	0.164	0.761	0.647
p-value	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001

^a–e There is a significant difference between the averages indicated by different letters of the same column. a: Readily degradable DM (%); b: degradable DM (%); c: degradation rate of DM (% h⁻¹); PD: potential degradability of DM (%) with a rumen outflow rate of 0.02; ED: effective degradability of DM (%) with a rumen outflow rate of 0.02 [39]; ¹ CON-DC: Control 1, alfalfa silage (40%) + dairy concentrate (60%) without additives; CON-DC: Control 1, alfalfa silage (40%) + dairy concentrate (60%) without additives; CON-FC: Control 2, alfalfa silage (20%) + fattening concentrate (80%) without additive; DCCEO: CON-DC + 60 mg/kg fresh mass of cinnamon essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of cinnamon essential oil; DCFEO: CON-DC + 60 mg/kg fresh mass of flaxseed essential oil; FCCEO: CON-FC + 60 mg/kg fresh mass of flaxseed essential oil; DCLEO: CON-DC + 60 mg/kg fresh mass of lemon seed essential oil; FCLEO: CON-FC + 60 mg/kg fresh mass of lemon seed essential oil. The abbreviations used in the tables are: DM for dry matter, OM for organic matter, CP for crude protein and NDF for neutral detergent fiber.

Other studies have reported varied impacts of EOs on digestion in vitro. For example, processing alfalfa silage with thyme EOs decreased in vitro digestion [8]. As a result of adding EOs, DM and OM digestibility decreased [40,41]. However, inoculation with EOs increased the digestibility of DM, OM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) [7]. Additionally, EOs affect degradability [15], cattle [3,34], and dairy cows [3]. The effect of EOs on rumen fermentation appears to depend on the type of diet consumed, as demonstrated in studies by Benchar et al. [3] and in vitro studies [13]. The variation in outcomes may be due to differences in the type, dose, chemical composition, conditions, and duration of the test period of the EO used in the studies.

3.3. Digestibility Parameters

The impact of the studied EOs on treatment digestibility is shown in Table 5. At a passage rate of 2% per h, FCLEO significantly enhanced the effective degradability (ED), as well as the soluble fraction of DM (a). Based on the results, the degradability potential (PD) of AS was found to be increased in both the FCLEO and DCLEO groups, in comparison to the CON group (p < 0.05). By adding DCLEO, the insoluble part of AS (b) decreased significantly in degradability in relation to the CON. As a function of the dose used at the silage level, the insoluble part degradability rate will increase or decrease. LEO (DC/FC) demonstrated a significant difference from the controls and was numerically higher than them at a constant DM degradation rate. In accordance with the reported results, a strong difference was found between the organic matter solution (a) and the control treatments (statistically significant). Compared to the CON, the FCCEO treatment significantly increased OM soluble fractions. DCCEO had the highest digestibility of OM (ED) at a passage rate of 2% per h. It appears that the rapidly decomposing part (a) of crude protein, FCLEO, has a significantly greater rate of degradability than the CON. Based on these results, it was found that EOs significantly reduced the insoluble fraction of CP (b) when added to the diet. With regards to the ED of crude alfalfa silage protein, the highest level was associated with the CON, and the lowest amount was associated with FCCEO/DCFEO, at 2% per hour, which demonstrates a significant decrease in ED when put in relation to all treatments (p < 0.05).

There are few studies on essential oils’ effects on rumen protein breakdown [24,42]. In a study by Molero et al. [17], the ruminal degradability of proteins in heifers was not significantly affected by the commercial component of EOs. Similarly, Benchar et al. [3] found that administering 2 g of commercial EO composition did not alter the degradability of protein in dairy cows’ rumens. However, there is evidence to suggest that EOs may affect the segregation of insoluble protein sources from soluble protein sources in the rumen, which can impact the binding and placement of plant materials entering the rumen. Additionally, a decrease in ruminal pH, as observed in cows fed concentrate-rich diets, can contribute to a decrease in degradability [43]. For some protein sources, inhibiting non-protein polymer hydrolysis, such as polysaccharides, may reduce the ability of proteolytic bacteria to access the substrate, leading to reduced protein degradability [44]. Furthermore, animals fed concentrate-rich diets have low activity of ruminal cellulolytic bacteria [45]. The main mechanism of action of EOs is to inhibit ammonia production from amino acids, according to Wallace and Cota [44]. EOs have this effect because they inhibit ammonia-producing bacteria. There was no inhibition of proteolytic and peptidolytic activities, confirming that deamination is the principal mechanism of action of EOs. Even though the overall effects may be lower than estimated, the authors hypothesize that a decrease in protein breakdown may also occur in addition to a reduction in deamination [46]. In the literature, the effects of EOs remain poorly understood [47]. Furthermore, EOs seem to affect ruminal protein breakdown differently depending on substrate type.

Therefore, it is recommended that further studies be conducted in vivo with cattle, sheep, and other important ruminants to validate the effects of EOs on animal performance. This includes nutrient utilization, feed efficiency, and animal health. In vivo studies can provide a more accurate assessment of the potential benefits and drawbacks of EOs in ruminant diets. Moreover, such studies can also help determine the optimal dosage of EOs and their conceivable interactions with other dietary components. This study gives valuable insights into the potential use of EOs as an alternative to traditional feed additives in ruminant nutrition.

4. Conclusions

Essential oils (EOs) have a substantial impact on in vitro fermentation, particularly lemon seed essential oil (LEO) at a concentration of 60 mg/kg of diet, which led to a reduction in gas formation in both dairy cattle and fattening diets. Furthermore, the addition of EOs to the diet significantly decreased the insoluble fraction of crude protein (CP) (b). These findings suggest that EOs have the potential to change the rumen’s microbial ecosystem and improve nutrient utilization in ruminants. However, it should be noted that in vitro studies have some limitations and may not necessarily reflect the in vivo effects of EOs on animal performance.