Materials and Methods

Plant Materials and Essential Oil Extraction

Aerial parts of P. abrotanoides Kar. (Figure 1) were collected at the flowering stage (late July 2023) from the mountainous regions of Arzaneh village, Bakharz County, Iran. The collection site was located at an altitude of 1550 m above sea level (34°57′27″ N, 60°10′05″ E), with clay‐loam soil. Plant samples were immediately transferred to the laboratory and shade‐dried at room temperature until a constant weight was reached. For essential oil extraction, a portion of the shade‐dried plant material was mechanically ground to pass through a 2‐mm sieve.

FIGURE 1: Whole plant of Perovskia abrotanoides Kar. at the flowering stage.

Essential oil extraction was performed using water distillation in a Clevenger‐type apparatus (Lenz Laborglasinstrumente GmbH, Wertheim, Germany) for 3 h. Approximately 100 g of air‐dried plant material was used for each distillation, and extraction was performed in triplicate to ensure reproducibility. The obtained essential oil was dried over anhydrous sodium sulphate to remove residual moisture, transferred into amber glass vials and stored at 4°C until analysis. Extraction yield was calculated as follows (Mahmodi et al. 2025):

Essentialoilyield%=Weightofessentialoilg/Dryweightofplantg×100

Chemical Characterization of the Essential Oil by GC–Flame Ionization Detector (FID) and GC/MS

Chemical composition of the essential oil was determined using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a FID and an Agilent 5975C mass selective detector (MSD). Separation was carried out on an HP‐5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; Agilent Technologies, Santa Clara, CA, USA). The oven temperature programme was as follows: initial 60°C for 2 min, then increased to 210°C at 3°C/min and finally to 240°C at 20°C/min, held for 8.5 min. Injector temperature was 240°C and detector temperature was 290°C for GC analysis. For GC/MS analysis, injector temperature was 280°C, ionization energy was 70 eV, and helium was used as carrier gas at a flow rate of 1 mL/min.

Identification of compounds was based on comparison of mass spectra with NIST and Wiley libraries, calculation of retention indices relative to n‐alkanes (C8–C24) and matching with reference data. Quantitative analysis was performed by area normalization without correction factors. Each essential oil sample was analysed in triplicate, and the average chromatographic peak areas were used for compound quantification.

Basal Diet and Rumen Fluid Collection

A basal diet (Table 1) containing 30% forage (alfalfa and clover) and 70% concentrate (barley, corn, soybean meal, sunflower meal, rice bran and sugar beet pulp) was formulated according to NRC (2007) recommendations for fattening sheep (30–40 kg body weight). This diet was not fed to the animals; rather, it was used as the substrate for in vitro incubations. The chemical composition (dry matter [DM] basis) was 16.6% crude protein, 2.28% ether extract, 4.53% ash, 48% non‐fibre carbohydrates, 1.1% Ca, 0.40% P and 2.35 Mcal/kg ME (Table 1).

Ingredients | % of DM
Dried alfalfa | 20
Clover forage | 10
Barley grain | 25
Corn grain | 15
Sunflower meal | 5
Sugar beet pulp | 5
Rice bran | 5
Soybean meal | 10
Vitamin–mineral supplementa | 2
Salt | 1
Limestone | 2
Chemical composition (% of DM)
Dry matter (% of fresh weight) | 81.2
Crude protein | 16.6
Ether extract | 2.28
Ash | 4.53
Non‐fibre carbohydrates | 48
Calcium | 1.1
Phosphorus | 0.4
Metabolizable energy (Mcal/kg DM) | 2.35

Prior to chemical analyses, feed samples were ground using a laboratory cyclone mill (Cyclotec 1093, Foss Tecator, Hillerød, Denmark) fitted with a 1‐mm sieve. The chemical composition of the basal diet components was determined according to the standard protocols of AOAC (2005). DM was determined by drying samples at 105°C for 2 h (Method 930.15). Crude protein (CP; N × 6.25) was determined using the micro‐Kjeldahl method (Method 984.13; Kjeltec 2300, Foss Tecator, Hillerød, Denmark). Ether extract (EE) was measured using an automated Soxhlet extraction apparatus (Soxtherm, C. Gerhardt GmbH & Co. KG, Königswinter, Germany) with petroleum ether (Method 920.39). Ash content was determined by incineration in a muffle furnace (Nabertherm GmbH, Lilienthal, Germany) at 600°C for 2 h (Method 942.05). Neutral detergent fibre (NDF) was analysed according to Van Soest et al. (1991) using heat‐stable alpha‐amylase and sodium sulphite and expressed inclusive of residual ash.

Rumen fluid was obtained before the morning feeding from three male Baluchi sheep (body weight: 30 ± 3.5 kg) surgically fitted with permanent rumen cannulas. Rumen contents were sampled from multiple sites (cranial, central and caudal regions) within the rumen using a flexible stomach tube connected to a manual vacuum suction pump. The collected contents were immediately filtered through four layers of cheesecloth into a pre‐warmed (39°C) insulated thermos flask that had been pre‐flushed with continuous CO2 to maintain strict anaerobic conditions during transfer to the laboratory. Donor animals were fed a maintenance diet consisting of wheat straw (200 g/d) and a commercial concentrate (300 g/d; 50% ground barley, 25% wheat bran, 20% soybean meal, 3% limestone, 1% vitamin–mineral premix, 1% salt) providing 10.15 MJ/kg DM.

In Vitro Gas Production and Nutrient Degradability

The experiment was conducted as a completely randomized design consisting of six treatments corresponding to six levels of P. abrotanoides essential oil (0, 100, 200, 300, 400 and 500 mg/L culture medium). Each treatment was represented by four incubation bottles (experimental units) per run, and the entire experiment was repeated in two independent incubation runs performed on different days using freshly collected rumen fluid. Therefore, a total of 48 incubation bottles (6 treatments × 4 replicates × 2 runs) were included in the experiment. Mean values obtained from the two incubation runs were used for statistical analysis to ensure analytical precision and reproducibility.

The artificial saliva (buffer solution) was prepared according to the standard protocol described by McDougall (1948). The buffer solution was maintained at 39°C and continuously saturated with CO2 until the pH stabilized at 6.8–6.9. Strained rumen fluid was then mixed with the artificial saliva buffer in a 1:2 (V/V) ratio under anaerobic conditions to prepare the final incubation medium. A 30 mL aliquot of this incubation medium along with 200 mg of the basal substrate (ground to pass through a 1‐mm sieve) was added to each bottle.

The bottles were sealed with rubber stoppers and aluminium caps and incubated at 39°C for 96 h. Incubation bottles were randomly assigned to incubation positions to minimize possible positional effects during incubation. Gas pressure and volume were recorded at 3, 6, 9, 12, 24, 48, 72 and 96 h according to the method of Theodorou et al. (1994) using a digital pressure transducer (Model PSA‐01‐RC1/8, Autonics, Busan, South Korea; measurement range: 0–100 kPa, accuracy: ±1% of full scale). Gas production data were fitted to the exponential model (Kazemi and Valizadeh 2023): where Y is the gas produced at time t (mL), b is the asymptotic gas production potential (mL), c is the fractional rate of gas production (h−1), and t is the incubation time (h).

Y=b1−e−ct

Post‐Incubation Sampling and Chemical Analyses

After 24 h of incubation, the pH was measured immediately using an electronic pH meter (Model HI2210‐01, Hanna Instruments, Woonsocket, RI, USA). Fermentation was stopped by placing the bottles on ice. The contents of each bottle were filtered through pre‐weighed polyester bags (pore size 45 µm) to separate the liquid and solid residues (Kazemi 2024).

The solid residue remaining in the bags was washed thoroughly with cold distilled water until the effluent ran clear and then dried at 60°C for 48 h to determine DM degradability. Subsequently, the dried residue was incinerated at 600°C for 2 h in a muffle furnace to determine organic matter degradability. The NDF degradability was evaluated by subjecting the un‐degraded solid residue to NDF analysis according to Van Soest et al. (1991) using heat‐stable α‐amylase.

For fluid analysis, total VFA concentration was determined by steam distillation using a Markham apparatus (Markham 1942). Methane volume was measured at 24 h by injecting 4 mL of 10 M NaOH into the headspace gas to absorb CO2, with the remaining gas considered as methane (Fievez et al. 2005). Ammonia nitrogen (NH3–N) concentration was determined by the micro‐Kjeldahl distillation method after mixing 5 mL of filtered culture fluid with 5 mL of 0.2 N HCl (AOAC 2005; Kazemi 2024). All analytical determinations were performed in duplicate.

Microbial Efficiency Indices

The partitioning factor (PF) and microbial mass yield (MMY) were calculated as (Kazemi and Saleh 2026) PF (mg truly digested OM/mL gas) = [Omi − (RDM − AshR)]/IVGPMMY (mg) = [OMi − (RDM − AshR)] − (IVGP × 2.2)where Omi is the initial organic matter (mg), RDM is the residual dry matter (mg), AshR is the ash in residual matter (mg), and IVGP is the net gas production at 24 h (mL).

Microbialsynthesisefficiency%=MMY/trulydigestedOM×100.

Total protozoa counts were determined using a Neubauer haemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda‐Königshofen, Germany) after fixing 5 mL of culture fluid with 5 mL of 50% formalin and staining with brilliant green (Dehority 2003).

Statistical Analysis

Data were analysed using the GLM procedure of SAS (Version 9.4; SAS Institute Inc., Cary, NC, USA) according to the following statistical model: where Yij is the observed response, μ is the overall mean, Ti is the fixed effect of essential oil level, and εij is the residual error.

Yij=μ+Ti+εij

Prior to analysis, the normality of residuals was evaluated using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene's test. When treatment effects were significant, means were compared using Tukey's multiple comparison test. In addition, orthogonal polynomial contrasts were performed to evaluate linear and quadratic responses to increasing essential oil supplementation levels. Statistical significance was declared at p < 0.05.