Results

Chemical Composition of Essential Oil

A total of 33 compounds were identified in the essential oil of P. abrotanoides, representing 99.4% of the total oil composition (Table 2). The essential oil yield was 1.5% (w/w) based on dry plant weight. The oil was dominated by oxygenated monoterpenes, with 1,8‐cineole (16.10%) and camphor (13.80%) accounting for nearly 30% of the total essential oil composition, followed by α‐pinene (8.40%), δ‐3‐carene (6.81%), n‐pentadecane (5.51%), (E)‐caryophyllene (4.21%) and α‐humulene (4.12%). The remaining constituents were present at concentrations below 4%.

No. | Chemical constituent | Amount (% W/W) | Retention index (RI)
1 | α‐Thujene | 0.40 | 924
2 | α‐Pinene | 8.4 | 932
3 | Camphene | 3.11 | 946
4 | Sabinene | 0.80 | 969
5 | β‐Pinene | 2.50 | 974
6 | Myrcene | 2.21 | 990
7 | δ‐3‐Carene | 6.81 | 1009
8 | α‐Terpinene | 1.30 | 1016
9 | p‐Cymene | 1.19 | 1024
10 | 1,8‐Cineole | 16.1 | 1031
11 | α‐Terpinolene | 2.12 | 1085
12 | Linalool | 2.53 | 1095
13 | Camphor | 13.8 | 1141
14 | α‐Terpineol | 2.81 | 1188
15 | Trans‐carveol | 2.52 | 1217
16 | Cis‐carveol | 0.40 | 1229
17 | Linalool acetate | 0.11 | 1257
18 | Bornyl acetate | 2.83 | 1285
19 | Carvacrol | 0.12 | 1300
20 | Methyl decanoate | 1.12 | 1321
21 | α‐Cubebene | 1.31 | 1371
22 | α‐Gurjunene | 0.34 | 1410
23 | (E)‐Caryophyllene | 4.21 | 1421
24 | α‐Humulene | 4.12 | 1455
25 | α‐Amorphene | 2.13 | 1481
26 | N‐pentadecane | 5.51 | 1500
27 | β‐Bisabolene | 0.21 | 1507
28 | γ‐Cadinene | 0.71 | 1514
29 | δ‐Cadinene | 2.51 | 1524
30 | Caryophyllene oxide | 0.45 | 1582
31 | β‐Cedrene epoxide | 1.51 | 1611
32 | Epi‐α‐cadinol | 2.23 | 1642
33 | (E,Z)‐farnesol | 2.01 | 1742

Gas Production Parameters and Methane Yield

Increasing levels of P. abrotanoides essential oil significantly reduced methane production by 57.5%, decreasing from 15.00 mL in the control treatment to 6.37 mL at 500 mg/L (linear effect, p < 0.0001) (Table 3). Likewise, gas production potential declined by 44.7%, whereas cumulative gas production after 24, 48, 72 and 96 h decreased by 47.9%, 47.0%, 43.7% and 44.2%, respectively, with increasing essential oil concentration (all linear effects, p < 0.0001). The fractional rate of gas production was also affected by treatment, with the lowest value observed at 500 mg/L (0.0848 h−1), corresponding to a 12.5% reduction compared with the control (cubic and quartic effects, p = 0.03). Significant quadratic responses were observed for gas production potential and cumulative gas production parameters (p = 0.0012–0.0030), whereas quartic effects were not significant.

Essential oil level (mg/L) | Methane1 (mL) | Fractional rate of gas production (h−1) | Gas production potential (mL) | 24‐h gas production (mL) | 48‐h gas production (mL) | 72‐h gas production (mL) | 96‐h gas production (mL)
0 (control) | 15.00a | 0.0969a | 72.23a | 59.32a | 69.80a | 74.17a | 74.95a
100 | 13.22b | 0.0920b | 61.34b | 49.83b | 59.18b | 62.95b | 63.33b
200 | 11.35c | 0.0993a | 56.08c | 46.00c | 54.22c | 57.87c | 58.32c
300 | 9.70d | 0.0978a | 50.08d | 40.35d | 48.27d | 51.97d | 52.22d
400 | 7.57e | 0.0965ab | 44.29e | 35.97e | 42.47e | 45.75e | 46.27e
500 | 6.37e | 0.0848c | 39.92f | 30.89f | 36.99f | 41.74f | 41.83f
SEM | 0.44 | 0.0012 | 2.26 | 0.66 | 0.64 | 0.69 | 0.70
p value
Linear | <0.0001 | 0.34 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001
Quadratic | 0.77 | 0.81 | 0.0012 | 0.003 | 0.002 | 0.0023 | 0.0010
Cubic | 0.79 | 0.03 | 0.0176 | 0.05 | 0.01 | 0.0084 | 0.0090
Quartic | 0.78 | 0.03 | 0.20 | 0.07 | 0.16 | 0.21 | 0.14

Protozoa Population and Fermentation Parameters

Increasing supplementation levels of P. abrotanoides essential oil resulted in a 33.8% reduction in total protozoal population, decreasing from 5.17 × 105 to 3.42 × 105 cells/mL (linear effect, p < 0.0001) (Table 4). Total VFA concentration decreased by 17.4%, whereas NH3–N concentration decreased by 29.4% relative to the control treatment (linear effect, p < 0.0001). In contrast, ruminal pH increased from 6.63 to 6.85, representing an increase of approximately 3.3%. No significant quadratic, cubic or quartic responses were detected for these variables (p > 0.05).

Essential oil level (mg/L) | Total protozoa (×105 cells/mL) | Total volatile fatty acid concentration (mmol/L) | NH3–N concentration (mg/dL) | pH
0 (control) | 5.17a | 96.00a | 24.25a | 6.63d
100 | 4.95a | 92.75a | 23.27a | 6.65cd
200 | 4.52b | 89.12b | 21.87b | 6.69c
300 | 4.22b | 85.62c | 19.75c | 6.77b
400 | 3.87c | 82.25cd | 18.62c | 6.80b
500 | 3.42d | 79.25d | 17.12d | 6.85a
SEM | 0.13 | 1.28 | 0.55 | 0.02
p value
Linear | <0.0001 | <0.0001 | <0.0001 | <0.0001
Quadratic | 0.76 | 0.98 | 0.54 | 0.34
Cubic | 0.66 | 0.89 | 0.31 | 0.28
Quartic | 0.58 | 0.96 | 0.58 | 0.39

Nutrient Degradability and Microbial Efficiency Indices

Supplementation with P. abrotanoides essential oil markedly improved microbial efficiency indices (Table 5). Microbial biomass production increased by 45.1%, microbial synthesis efficiency increased by 79.6% and PF increased by 55.3% compared with the control treatment (all linear effects, p < 0.0001). Conversely, NDF degradability, OM degradability and DM degradability decreased by 19.4%, 14.0% and 11.9%, respectively, as essential oil concentration increased. A significant quadratic effect was observed only for DM degradability (p = 0.025), whereas no other quadratic, cubic or quartic effects were detected (p > 0.05).

Essential oil level (mg/L) | Microbial biomass production (mg) | Microbial biomass synthesis efficiency (%) | Partitioning factor (mg truly degraded organic matter/mL gas produced) | Neutral detergent fibre degradability (%) | Organic matter degradability (%) | Dry matter degradability (%)
0 (control) | 58.32d | 30.87f | 3.18e | 50.25a | 75.01a | 73.75a
100 | 63.46c | 36.67e | 3.47d | 48.50a | 73.01ab | 71.25b
200 | 66.88c | 39.80d | 3.65d | 46.50b | 70.50b | 67.50c
300 | 75.06b | 45.81c | 4.06c | 44.75b | 67.50c | 67.25cd
400 | 76.44b | 49.09b | 4.33b | 42.50c | 67.25c | 66.50cd
500 | 84.63a | 55.46a | 4.94a | 40.50d | 64.50c | 65.00d
SEM | 1.93 | 1.72 | 0.12 | 0.74 | 0.83 | 0.68
p value
Linear | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001
Quadratic | 0.65 | 0.48 | 0.47 | 0.76 | 0.42 | 0.025
Cubic | 0.34 | 0.98 | 0.91 | 0.91 | 0.31 | 0.76
Quartic | 0.20 | 0.11 | 0.24 | 0.82 | 0.70 | 0.19