Home Life Sciences Testing thawed rumen fluid to assess in vitro degradability and its link to phytochemical and fibre contents in selected herbs and spices
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Testing thawed rumen fluid to assess in vitro degradability and its link to phytochemical and fibre contents in selected herbs and spices

  • Kawa Merkhan ORCID logo EMAIL logo , James Standen ORCID logo and Abdul Shakoor Chaudhry ORCID logo
Published/Copyright: November 4, 2025
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Open Agriculture
From the journal Open Agriculture Volume 10 Issue 1

Abstract

This study used thawed rumen fluid (TRF) to assess in vitro degradability and its relationship with the phytochemical and fibre contents of four herbs (green tea leaves, great burnet leaves, eucalyptus leaves, and oregano leaves) and five spices (black seed, cumin seeds, garlic bulb, onion flesh, and grape peel) using multivariate approaches. Duplicate samples of each herb and spice were incubated with TRF from each of four replicated steers for 48 h in an ANKOM DaisyII incubator. The results showed that each group of herbs and spices had different proximate, fibre, and phytochemical contents. Apparently, TRF was effective in estimating the in vitro degradability of different herbs and spices. Moreover, in vitro degradability was positively associated with total saponin content, while negatively correlated with fibre fractions. Principal component analysis identified two main dimensions, one associated with ‘fibre fractions’ and the other with ‘phytochemicals’, which were interpreted as the main factors influencing degradability. The multiple regression analysis demonstrated a positive correlation coefficient for the phytochemical contents of garlic bulb and onion flesh, indicating a considerable improvement in dry matter degradability (DMD). Additionally, the DMD values were significantly improved, as indicated by the positive correlations for the fibre fractions of onion flesh and green tea leaves. It can be concluded that the current multivariate analysis may be more accurate and useful for selecting or ranking various plants before their use as feed additives. However, further in vitro studies are needed to examine the effects of different levels of herbs and spices on degradability, fermentation, and gas production profiles of a much wider range of feeds and forages. This could be achieved by using TRF when fresh rumen fluid is not easily available due to the ever-increasing restrictions and logistics at an abattoir.

Keywords: thawed rumen fluid; fibre; tannin; saponin; degradability; covariate

1 Introduction

Conducting animal-based in vivo experiments is challenging, invasive and costly, as it requires large amounts of different feeds to determine the degradability and digestibility of ingredients [1]. Additionally, animal trials are often inappropriate for evaluating single feedstuffs [2]. As a result, various in vitro methods using fresh [3], [4], [5], [6] or thawed [7], [8], [9], [10] rumen fluid have been developed as inexpensive alternatives to assess the nutritional value of feeds [11].

The nutritional value of a ruminant feed is determined not only by its chemical composition but also by the efficiency of its microbial degradation and digestion [12]. Plant-derived products and essential oils are increasingly investigated for their potential to improve nutrient use efficiency and reduce environmental impact [13]. This is linked to their secondary metabolite contents, such as saponins and tannins, which can not only exert anti- or pro-bacterial effects [14], but also contribute protein, fibre, and minerals that are found in most plants, such as tea leaves [15].

Different plants vary considerably in their content of phytochemicals and nutrients. For example, Sanguisorba officinalis is rich in phenolics, polysaccharide, and tannins with strong antioxidant properties [16]; black seed has been described as a ‘miracle plant’ due to its antibacterial activities [17]; eucalyptus leaves contain flavonoids and phenols [18]; garlic and onions are rich in saponins [19], and organosulfur compounds as the dominant bioactive constituents [20]; and grape pomace [21] and green tea [22] contain high concentrations of tannins. Understanding the phytochemical and proximate composition of herbs and spices is therefore essential, since excessive intake of some metabolites may limit their value as feed additives.

Despite their potential, the effects of plant secondary metabolites on ruminant fermentation remain inconsistent, partly due to variability in composition and interactions with other feed components such as fibre [23]. Rumen modifiers (e.g., essential oils, tannins, saponins, lipids) are widely available, but their efficacy is inconsistent across studies [24]. This makes evaluating different plant parts and their combined effects on degradability a continuing research priority.

In vitro fermentation studies typically rely on fresh rumen fluid (FRF) as an inoculum source, which requires immediate handling and continuous access to donor animals. However, obtaining FRF presents logistical and ethical challenges, particularly in facilities without in-house ruminants. Thawed rumen fluid (TRF) has emerged as a practical alternative, with recent studies showing comparable fermentation patterns to FRF under controlled conditions [7], [8], [9, 22], 25]. In the present study, TRF was utilised to assess the degradability of various herbs and spices. Multivariate techniques were further applied to link degradability outcomes with biochemical composition, thereby supporting a more informed selection of herbs and spices as potential ruminant feed additives.

2 Materials and methods

2.1 Ethics statement

The studies were approved by the Animal Welfare and Ethical Review Board (AWERB) of Newcastle University, UK (Project ID No: 1089). Instead of using live and surgically modified animals, only steers that were freshly slaughtered for food were used to obtain rumen fluid (RF). Therefore, ethical standards for the use of animal-derived materials from only the UK approved farms were followed in this study.

2.2 Experimental design

In vitro incubation was conducted using a DAISYII incubator with four jars, each inoculated with buffered rumen fluid from one steer, effectively simulating four steers as biological replicates. While initial tests revealed no significant steer effect (P > 0.05), these individual jars were still treated as biological replicates to ensure statistical robustness and cover for inherent biological variability. Therefore, each plant material was tested in duplicate within each of four jars, resulting in eight replicated observations per treatment for each herb or spice.

The study was conducted using a completely randomised experimental design, with TRF from four replicated steers and duplicate sample bags in the ANKOM Daisy incubator. The experiment tested the in vitro DMD (IVDMD) and organic matter degradability (IVOMD) of four herbs: green tea leaves (Camellia sinensis, GTL), great burnet leaves (S. officinalis, GBL), oregano leaves (Origanum vulgare, OL), and eucalyptus leaves (Eucalyptus globulus, EL), as well as five spices: black seeds (Nigella sativa, BS), cumin seeds (Cuminum cyminum, CS), garlic bulbs (Allium sativum, GB), onion flesh (Allium cepa, OF), and grape peel (Vitis vinifera, GrP) before their use as feed additives.

2.3 Collection and storage of rumen fluid

Representative samples of frozen RF, stored at −20 °C for up to one month, were used in this study. These frozen samples were preserved in the laboratory from their fresh counterparts, which were obtained from four humanely killed steers at a local abattoir. The steers represented different breeds, ages, and farm locations, as shown in Table 1. Before slaughter, they had been fed diets containing fresh, dried, and ensiled grass with concentrates. This reflects expected variations amongst animals that are routinely received and processed at a typical abattoir.

Table 1:

Pre-slaughter features of four steers used to collect rumen fluid for in vitro incubations.

Parameters Steers
1 2 3 4
Farm location Morpeth, Northumberland Wigton, Cumbria Thornton, Bradford Morpeth, Northumberland
Breed Aberdeen Angus X Ayrshire X Limousin X Aberdeen Angus X
Age in days 591 493 863 724

Soon after collection, the RF was filtered through four layers of muslin cloth using a large funnel and immediately transferred into pre-warmed, sterile, insulated thermos flasks (Thermos Ltd, UK) maintained at around 39 °C, which were flushed with CO2 and screw capped to maintain anaerobic conditions. The filtered samples were then transported to the laboratory for use as FRF in another in vitro study, and two 2 mL samples were each acidified at a 1:1 ratio with 1 N hydrochloric acid (HCl) and stored at −20 °C for subsequent analysis of ammonia nitrogen (NH3–N) and volatile fatty acids (VFAs). Simultaneously, measured portions of FRF from each of the four steers were aliquoted into sterile containers, flushed with CO2 to remove residual O2, tightly sealed, and stored at −20 °C until further use. Before incubation, the samples were thawed at 40 °C in a water bath and then filtered again through muslin cloth to remove large residual particulates and obtain TRF. Before incubation with the test feedstuffs, TRF from each steer was incubated under standard in vitro conditions without substrate to assess baseline fermentation traits, including pH, NH3–N, and VFAs. These values were compared with FRF from our lab and published data [2], 6], 10], 25] to confirm that TRF retained comparable fermentation characteristics, ensuring its suitability for ranking feeds in subsequent incubations. Although FRF is preferred, previous studies [4], [19], [20], [21], [22] have shown that properly handled TRF could still provide comparable, albeit lower, fermentation characteristics for ranking feeds. Although rumen fluid was collected in a single event on the same day, the samples were obtained from four different steers representing variation in breed, age, and diet. This approach introduces biological variability, while temporal variation across multiple collections is acknowledged as a limitation, but consistent with the previous reports [5], [7], [8], [9, 25].

2.4 Buffered inoculum

McDougall’s synthetic saliva [26] (Supplementary Table S1) was used as a buffer solution. It was placed into dark bottles, flushed with CO2, screw-capped, and maintained in a water bath at 39 °C. The frozen RF at −20 °C was thawed in a water bath at approximately 40 °C until fully liquefied, then filtered through four layers of muslin cloth to obtain TRF. Approximately 400 mL of TRF from each steer was then mixed with 1,600 mL of buffer in each 2.5 L Daisy jar, simulating a rumen environment. The jars were purged with CO2 to displace oxygen and firmly closed.

For subsequent analysis of NH3–N and VFAs, two 2 mL aliquots of TRF were each acidified at a 1:1 ratio with 1 N HCl and stored at −20 °C.

2.5 In vitro degradation using the ANKOM DAISYII incubator

The DaisyII incubator (ANKOM Technology) consisted of four separate digestion jars that allowed for constant rotation of the fermentation medium at the required temperature (39.5 ± 0.5 °C). Each jar acted as a simulated steer as it contained buffered TRF from a single steer as a biological replicate. For each jar, duplicate powder samples from each herb and spice were individually weighed into fibre filter bags (F57, pore size 25 µm; Ankom Technology®, Macedon, NY, USA), which were then sealed and incubated. The method followed standard procedures according to the operating instructions provided by the manufacturer (Ankom Technology®, Macedon, NY, USA).

Ground samples (0.50 g per bag) of each herb and spice were weighed into duplicate fibre filter bags, with two empty bags serving as blanks. The bags were heat-sealed and placed in digestion jars containing the buffered TRF. Although each sample was incubated in a separate bag within the same jar, we acknowledge that incubating multiple treatments together could allow cross-treatment interactions, for example, tannins from high-tannin substrates potentially affecting microbial activity in other samples. This limitation is inherent to the batch incubation system. However, using a shared environment mimics the natural rumen of an intact or a fistulated animal, where multiple feed components are fermented simultaneously. This approach ensures that all samples experienced identical fermentation conditions, reducing within-jar variability.

At the end of the 48-h incubation period, the jars were removed from the DaisyII chamber. The bags were collected, rinsed with tap water, oven-dried (Genlab drying oven, England) at 60 °C for 72 h, and weighed to determine IVDMD. Then the dried residues were ignited in a furnace at 550 °C for 5 h to determine organic matter (OM) content before estimating IVOMD.

2.6 Processing and biochemical analysis of samples

Before chemical analysis, all herb and spice samples were ground through a 1 mm sieve using a sample mill (TWISTER – Cyclone Mill 20.831, Germany). Dry matter (DM), organic matter (OM), and ether extract (EE) were determined using standard techniques [27], while crude protein (CP) was assessed by measuring total nitrogen (N) (N × 6.25 = CP) using a combustion assay (Elemental analyser, LECO CHN628, USA). The neutral detergent fibre (NDF) of each sample was determined using the previously reported method [28], without the use of amylase, dekalin, and sodium sulfite. The acid detergent fibre (ADF) and acid detergent lignin (ADL) contents were also determined [29]. The cellulose content was calculated by subtracting ADL values from ADF values, while the hemicellulose content was calculated by subtracting ADF values from NDF.

Total tannins (TT) were estimated using the Folin-Ciocalteu method [30], with tannic acid (Fisher Scientific, UK) as the standard. The condensed tannin (CT) content was determined using the butanol-HCl method [30], with (−)-epigallocatechin gallate (eMolecules, Fisher Scientific, UK) as the standard. The vanillin/HCl method [31] was employed to determine the total saponin (TS) content, using diosgenin (Fisher Scientific, UK) as the reference standard. A UV-VIS spectrophotometer (Libra S12, Biochrom Ltd, Cambridge, UK) was used to measure absorbance at 725 nm, 400 nm, and 544 nm for analysing TT, CT, and TS, respectively.

Ammonia nitrogen (NH3–N) was analysed according to the method of Broderick and Kang [32], using NH4Cl in H2O as the ammonium standard solution. A UV-VIS spectrophotometer (Libra S12, Biochrom Ltd, Cambridge, UK) was used to measure and record absorbance at 550 nm.

Volatile fatty acids, including acetate, propionate, n-butyrate, n-valerate, isobutyrate, and isovalerate, were analysed using a Dionex Aquion Ion Chromatography System (Thermo Scientific™, UK) equipped with an AS-AP autosampler and a Dionex IonPac ICE-AS1 column (4 × 250 mm). A 25 µL injection was run with a 1 mM heptafluorobutyric acid eluent at an isocratic flow rate of 0.16 mL/min for 35 min. The ACRS-ICE500 suppressor was used with 5 mM tetrabutyl ammonium hydroxide. The column oven temperature was set at 30 °C. Analytical-grade standards of acetic, propionic, butyric, valeric, isobutyric, and isovaleric acids were run at 5, 50, and 100 ppm. Total VFA concentration was calculated as the sum of all quantified acids.

2.7 Statistical analysis

All data sets were arranged in Microsoft Excel data sheets for Microsoft 365 (Part of Office 365 ProPlus, Newcastle University) before applying suitable comparative or statistical analyses by using Minitab 21 software as described below:

One-way ANOVA tests were conducted to compare the fermentation profiles of FRF with TRF after one month of freezing, with significance set at P < 0.05. Additionally, qualitative assessments compared the effectiveness of TRF with FRF based on previous studies from the same laboratory and from other research groups.

Moreover, one-way ANOVA was used to compare different materials within each group of herbs or spices for different proximate and phytochemical contents. Tukey’s post hoc test was employed to compare the means of each content within each group of herbs and spices (P < 0.05). Additionally, the effect of each herb and spice on in vitro degradability was examined using one-way ANOVA across various herbs and spices. Tukey’s post hoc test was also used to evaluate the individual means of IVDMD and IVOMD, among GTL, EL, OL, GBL, BS, CS, GB, OF, and GrP at P < 0.05.

Pearson’s correlation was used to explore relationships between each phytochemical or fibre fraction and each of the IVDMD and IVOMD of the tested herbs and spices. Only variables that showed significant correlations were further analysed using polynomial regression.

Principal component analysis (PCA) was used to examine patterns and relationships among different biochemical parameters. This multivariate method helps reduce data complexity while retaining essential information. Rotated factor loadings and commonalities were calculated to better understand how well each variable was represented by the extracted components.

Multiple regression analysis was then applied to determine how phytochemical compounds and fibre fractions influenced IVDMD. Additional regression models were developed using the first two principal components to assess their effect on IVDMD, based on the proportion of total variance they explained. The analysis focused on key variables including TT, CT, TS, and fibre fractions (NDF, ADF, and ADL).

3 Results

3.1 Comparative evaluation of thawed rumen fluid

In the current study, TRF displayed fermentation characteristics that were largely comparable to the FRF under identical conditions (Table 2). No differences (P > 0.05) were found between TRF and FRF in pH, NH3–N, total VFA, or main VFA components, including acetate, propionate, butyrate, and isovalerate. However, a slight but statistically significant difference was observed in valerate (P < 0.05), and a highly significant difference was noted in isobutyrate concentrations (P < 0.01), with TRF showing increased levels than FRF.

Table 2:

Comparison of fermentation characteristics between fresh and thawed rumen fluid from the current study and reference values reported in this and other laboratories.

Traits Current study RF from literature
FRF TRF SEM This lab FRF [3] Other labs [6], 10], 25]
FRF TRF
pH 7.5 7.5 0.19NS 6.8–7.3 6.4–6.9 6.8–6.9
NH3–N mg/L 195 127 20.4NS 63–123 134–261 64–127
Total VFAs 86.3 93.8 6.97NS 61–82 47–122 43–64
VFAs (mmol/L)
Acetate 53 64 5.05NS 53–76 30–77 27–29
Propionate 24 21 2.4NS 18–31 9–15 7–10
Butyrate 8 6 0.8NS 4.4–8.5 6–13 4–6
Valerate 0.8 0.4 0.13* 0.4–1.4 0.6–1.3 0.5–0.7
Isobutyrate 0.7 1.5 0.18** 0.7–2.6 0.4–13 0.3–23
Isovalerate 1.1 1.0 0.28NS 0.9–3.8 0.8–1.7 0.6–0.8

  1. RF, rumen fluid; FRF, fresh rumen fluid; TRF, thawed rumen fluid; NH3–N, Ammonia concentration; VFAs, volatile fatty acids; n.d., not detected; SEM, standard error of the mean; P < 0.05 (*), P < 0.01 (**), NS, not significant.

3.2 Proximate analysis

Table 3 shows differences between each set of herbs and spices for different proximate analyses (P < 0.01). Among the herbs, the CP contents of GBL and GTL were comparable and higher than those of the other herbs. Meanwhile, EL exhibited greater levels of EE, ADF, and ADL compared to the other herbs. Additionally, NDF and cellulose contents were higher in OL than in the other herbs. The hemicellulose contents of GTL and OL were comparable and significantly higher than those of GBL and EL. Regarding spices, BS had a significantly higher EE content than the other spices. The CP contents of BS and GB were similar and considerably higher than those of the other spices. Furthermore, GrP showed greater levels of NDF, ADF, and ADL than the other spices, while the hemicellulose content of GB was lower than that of BS, CS, and GrP.

Table 3:

Mean (n = 3) proximate composition (g/kg DM), standard error of means (SEM) and significance levels for values for four herbs and five spices.

Herb/spice Proximate content
DM OM CP EE NDF ADF ADL Hemicellulose Cellulose
Herbs
GBL 951B 897D 258A 40B 202D 164C 146C 37b 17B
EL 949C 957A 63C 118A 353B 330A 291A 22b 38A
GTL 965A 953B 256A 44B 251C 137C 127C 110a 10B
OL 964A 926C 92B 79AB 391A 279B 239B 107a 40A
SEM 2.17*** 7.28*** 27.3*** 11.5* 23.0*** 24.2*** 20.5*** 12.6*** 4.18***
Spices
BS 975A 960B 198A 315A 362C 182B 132B 175a 50A
CS 969B 886E 170B 79B 406B 222B 184B 179a 38A
GB 969B 969A 186A 9B 14E 5C n.d. 8b n.d.
OF 924C 930D 125C 28B 138D 173B 153B n.d. 19A
GrP 969B 937C 138C 60B 548A 432A 405A 175a 27A
SEM 4.99*** 7.82*** 7.52*** 30.5*** 51.4*** 37.0*** 34.1*** 22.2*** 5.79NS

  1. GBL, great burnet leaf; EL, eucalyptus leaf; GTL, green tea leaf; OL, oregano leaf; BS, black seed; CS, cumin seed; GB, garlic bulb; OF, onion flesh; GrP, grape peel; DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fibre; ADF, Acid detergent fibre; ADL, acid detergent lignin. Means within a column for each nutrient in each group of herbs or spices with different letters (A, B, C and D) differed significantly (P < 0.05). Here *, *** and NS represent either significance at P < 0.05 and P < 0.001, or non-significant, respectively.

3.3 Phytochemical contents

The main differences for mean phytochemical amounts between various herbs and spices are shown in Table 4. The results for herbs indicated that the TT and CT contents of GTL, GBL, and EL were higher than those of OL. Great burnet leaves, on the other hand, contain significantly more TS than the other herbs. In terms of spices, the levels of TT and CT in grape peel were higher than those in the other spices. Additionally, GB and OF contain considerably more TS than the other spices.

Table 4:

Mean (n = 3) phytochemical contents (g/kg DM), standard error of means (SEM) and significance levels for values for herbs and spices.

Herb/spice Secondary metabolites content
Total tannin Condensed tannin Total saponin
Herbs
 GBL 69A 25A 11.8A
 EL 65A 30A 8.0B
 GTL 79A 28.4A 4.1C
 OL 20B 2.4B 8.0B
SEM 7.68*** 3.39*** 0.88***
Spices
 BS 3.68B 0.51B 11.89BC
 CS 3.82B 0.88B 13.45B
 GB 1.39B 0.98B 35.05A
 OF 0.73B 0.27B 33.44A
 GrP 8.44A 4.78A 10.32C
SEM 0.77*** 0.45*** 2.95***

  1. GBL, great burnet leaf; EL, eucalyptus leaf; GTL, green tea leaf; OL, oregano leaf; BS, black seed; CS, cumin powder; GB, garlic bulb; OF, onion flesh; GrP, grape peel. Means within a column for each content in each group of herbs or spices with different letters (A, B, C and D) differed significantly (P < 0.05). Here *** represents significance at P < 0.001.

3.4 In vitro degradability

Table 5 presents the mean in vitro degradability values of various herbs and spices. Different herbs and spices showed significant differences (P < 0.001) in their IVDMD and IVOMD. The highest IVDMD and IVOMD were observed in GB and OF, followed by GTL, CS, OL, GBL, BS, EL, and GrP.

Table 5:

Mean (n = 8) in vitro dry matter and organic matter degradability (g/kg DM) using the ANKOM DaisyII system of different herbs and spices.

Herb/spice Degradability (g/Kg DM)
Dry matter Organic matter
Great burnet leaves 525CD 554D
Eucalyptus leaves 420E 461E
Green tea leaves 687B 702B
Oregano leaves 563C 604CD
Black seed 475DE 518DE
Cumin seed 635B 653BC
Garlic bulb 996A 996A
Onion flesh 949A 968A
Grape peel 329F 335F
SEM with significance 26.0*** 36.7***

  1. Means within a column with different letters differ significantly (P < 0.01). Mean values were significantly different at P < 0.001 (***); SEM, standard error of the means; n, number of replicates.

3.5 Relationship between degradability and phytochemical contents and fibre fractions of herbs/spices

3.5.1 Correlation coefficient

The Pearson correlation coefficients between the phytochemical contents, fibre fractions of all herbs and spices, and IVDMD and IVOMD are shown in Table 6. A strong positive association (P < 0.01) was found between TS content in herbs and spices and degradability. The correlation coefficients (r) between TS and IVDMD and IVOMD were 0.82 and 0.80, respectively. Additionally, IVDMD and IVOMD showed a significant negative correlation (P < 0.01) with NDF, ADF, and ADL contents. The correlation coefficients between NDF and IVDMD and IVOMD were −0.85 and −0.87, respectively. For ADF, the correlation coefficients were −0.78 and −0.80, respectively, while the correlation values between ADL and IVDMD and IVOMD were −0.61 and −0.65, respectively. In contrast, the correlation coefficients between IVDMD and IVOMD with TT, CT, hemicellulose, and cellulose were negative but not statistically differ (Table 6). Furthermore, Table 7 shows the results of a polynomial regression analysis to relate the significant contents like TS, NDF, and ADF to the DM and OM degradability of the herbs and spices.

Table 6:

Correlation coefficients (r) between in vitro degradability traits and phytochemical and fibre contents of herbs and spices.

Parameter IVDMD IVOMD
Total tannin −0.25ns −0.25ns
Condensed tannin −0.29ns −0.29ns
Total saponin 0.82** 0.80**
NDF −0.85** −0.87**
ADF −0.78** −0.80**
ADL −0.61** −0.65**
Hemicellulose −0.29ns −0.28ns
Cellulose −0.32ns −0.28ns

  1. IVDMD, in vitro dry matter degradability; IVOMD, in vitro organic matter degradability; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; **, P < 0.01; ns, non-significant.

Table 7:

Model performance metrics (standard error, R-squared, and P-values) for predicting IVDMD and IVOMD from total saponin (TS), neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL) using linear, quadratic, and cubic regression.

Response Predictor Standard error of the regression (S) R-squared (%) P-value
Linear Quadratic Cubic
IVDMD TS 93.6 85 0.0001 0.001 0.008
IVOMD TS 89.99 85 0.0001 0.0001 0.014
IVDMD NDF 121.7 74 0.0001 0.0262 0.615
IVOMD NDF 110.9 76 0.0001 0.381 0.431
IVDMD ADF 146.7 62 0.0001 0.333 0.850
IVOMD ADF 133.4 66 0.0001 0.362 0.803
IVDMD ADL 151.4 46 0.002 0.921 0.084
IVOMD ADL 139.7 49 0.001 0.944 0.129

  1. IVDMD, in vitro dry matter degradability; IVOMD, in vitro organic matter degradability; TS, total saponin; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin.

3.5.2 Principal components analysis (PCA)

Table 8 and Figure 1 show the Principal Component Analysis results indicating the dataset’s underlying structure. It appears that only the first three components were determined to be relevant, accounting for 38.7 %, 33.3 %, and 20.2 % of the total variance among the seven investigated features. The fibre contents – specifically NDF, ADF, and ADL – loaded significantly on the first factor, with positive loadings of 0.81, 0.97, and 0.99, respectively. So, this component was referred to as the “fibre fraction” factor. The second component, derived from the phytochemical contents, exhibited strong negative loading scores for TT (−0.92) and CT (−0.91), while TS had a positive loading score (0.79); thus, it may be defined as the “phytochemical content” factor. Cellulose was found to load negatively on the third component with a score of −0.90, which is interpreted as the “cellulose” factor and represents a unique aspect of the dataset.

Table 8:

Rotated factor loadings and final communality estimates (c) from principal components analysis on phytochemical and fibre contents of herbs/spices.

Traits Component (c)a
1 2 3
Total tannin −0.18 −0.92 0.28 0.96
Condensed tannin −0.09 −0.91 0.31 0.93
Total saponin −0.30 0.79 0.41 0.88
Neutral detergent fibre 0.81 0.02 −0.50 0.91
Acid detergent fibre 0.97 0.05 −0.12 0.96
Acid detergent lignin 0.99 0.01 0.05 0.98
Cellulose 0.04 0.18 −0.90 0.85
Total
Variance 2.707 2.332 1.416 6.46
Variance explained (% total) 38.7 33.3 20.2 92

  1. a(c), Final communality estimates. The bold values indicate the highest loading scores for each component.

Figure 1: Biplot of the first two principal component (PC) score vectors, describing the classification of each phytochemical and fibre content within the PC loading vectors. PC, principal component; TT, total tannin; CT, condensed tannin; TS, total saponin; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin.
Figure 1:

Biplot of the first two principal component (PC) score vectors, describing the classification of each phytochemical and fibre content within the PC loading vectors. PC, principal component; TT, total tannin; CT, condensed tannin; TS, total saponin; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin.

The PCA results identified two meaningful components: (1) a ‘fibre fraction’ factor, capturing variance in NDF, ADF, and ADL, and (2) a ‘phytochemical content’ factor, predominantly influenced by tannins and saponins. Although the first two principal components explained 72 % of the total variance, PCA still provided meaningful insights into the clustering of samples based on their chemical profiles and degradability characteristics. The overlap among herbs and spices in PC space suggests some degree of compositional similarity, particularly in fibre and phytochemical content. Notably, samples with high tannin or saponin contents tended to cluster away from those with lower levels, offering preliminary differentiation that can inform subsequent feed additive screening and ranking.

3.5.3 Multiple regression of PCA and IVDMD results

Multiple regression analysis was used to investigate the complex correlations between independent variables, specifically the phytochemical content factor (TT, CT, and TS) and the fibre fraction factor (NDF, ADF, and ADL), and the dependent variable, IVDMD (Tables 9 and 10). The analysis focused on small variations among nine different herbs and spices: GTL, GBL, EL, OL, BS, CS, GB, OF, and GrP. Each herb and spice was examined separately for its effect on IVDMD, without a specified reference herb or spice. The coefficients indicate the average difference relative to the overall mean of the dependent variable.

Table 9:

Multiple regression coefficients for IVDMD (g/kg) across categorical (herbs and spices) and continuous (TT, CT, and TS g/kg DM) variables (R 2 = 99.49 %).

Term Coef SE coef t-Value P-value
Constant 556.6 90.4 6.16 0.001
 TT 1.154 0.620 1.86 0.082
 CT −3.22 4.58 −0.70 0.493
 TS 4.38 4.67 0.94 0.363
Herbs/spices
 GBL −72.1 59.1 −1.22 0.241
 BS −154.5 47.6 −3.25 0.005
 CS 35.4 42.7 0.83 0.421
 EL −141.1 79.5 −1.78 0.096
 GB 286.7 92.6 3.10 0.007
 GrP −275.9 36.6 −7.54 0.001
 GTL 119.6 79.4 1.51 0.153
 OL −64.1 55.9 −1.15 0.269
 OF 266.2 86.2 3.09 0.008

  1. Coef, coefficient; SE, standard error; TT, total tannin; CT, condensed tannin; TS, total saponin; GBL, great burnet leaves; BS, black seeds; CS, cumin seeds; EL, eucalyptus leaves; GB, garlic bulb; GrP, grape peel; GTL, green tea leaves; OL, oregano leaves; OF, onion flesh.

Table 10:

Multiple regression coefficients for IVDMD g/kg across categorical (herbs and spices) and continuous (NDF, ADF, and ADL g/kg DM) variables (R 2 = 99.12 %).

Term Coef SE coef t-Value P-value
Constant 211 225 0.94 0.365
 NDF 0.991 0.637 1.56 0.144
 ADF 0.515 0.484 1.06 0.307
 ADL −0.419 0.430 −0.97 0.348
Herbs/spices
 GBL 101.0 87.3 1.16 0.268
 BS −151.3 29.0 −5.22 0.001
 CS 1.5 50.0 0.03 0.976
 EL −179.1 30.3 −5.91 0.001
 GrP −486 150 −3.25 0.006
 GTL 216.6 61.2 3.54 0.004
 OL −99.3 42.3 −2.35 0.035
 OF 597 127 4.71 0.001

  1. Coef, coefficient; SE, standard error; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; GBL, great burnet leaves; BS, black seeds; CS, cumin seeds; EL, eucalyptus leaves; GrP, grape peel; GTL, green tea leaves; OL, oregano leaves; OF, onion flesh.

The multiple regression analysis explored various predictors of IVDMD across different herbs and spices. Among the independent variables examined, TT, CT, and TS did not demonstrate significant relationships with IVDMD (TT: coef. = 1.15, P = 0.08; CT: coef. = −3.22, P = 0.49; TS: coef. = 4.38, P = 0.36). These findings suggest that variations in TT, CT, and TS contents may not reliably predict changes in IVDMD across different categories of herbs and spices. Conversely, GB (coef. = 287, P = 0.007) and OF (coef. = 266, P = 0.008) showed significant positive associations, indicating that they enhance IVDMD. Additionally, variables such as GrP (coef. = −276, P = 0.001) and BS (coef. = −155, P = 0.005) exhibited significant negative relationships with IVDMD, suggesting that higher levels of BS and GrP are associated with reduced IVDMD. Meanwhile, GBL, GTL, OL, EL, and CS did not show significant relationships.

Among the independent variables examined, the NDF, ADF, and ADL contents did not demonstrate significant relationships with IVDMD (NDF: coef. = 0.99, P = 0.14; ADF: coef. = 0.52, P = 0.31; ADL: coef. = −0.42, P = 0.35). Conversely, OF (coef. = 597, P = 0.001) and GTL (coef. = 217, P = 0.004) showed significant positive associations, indicating that they enhanced IVDMD. Additionally, variables such as GrP (coef. = −486, P = 0.006), EL (coef. = −179, P = 0.001), BS (coef. = −151, P = 0.001), and OL (coef. = −99, P = 0.04) exhibited significant negative relationships with IVDMD, suggesting that higher levels of GrP, EL, BS, and OL were associated with reduced IVDMD. Meanwhile, GBL and CS did not show significant relationships with IVDMD.

4 Discussion

Evaluation of the functional properties of herbs and spices, including proximate and secondary metabolite compositions, is essential. Many plants are consumed without adequate information about their metabolites, which, if ingested in excess, may limit their effectiveness as feed additives. Therefore, the primary aim of this study was to assess selected herbs and spices for their proximate and phytochemical contents, as well as their in vitro degradability, before considering them as potential ruminant feed additives.

Recent research has supported using TRF as an inoculum source for in vitro fermentation studies. Recently, Ma et al. [5] demonstrated that the concentrate-to-forage ratio affects fermentation and bacterial diversity independently of inoculum type, confirming the suitability of TRF for evaluating starch content effects. Similarly, Tunkala et al. [9], 33] showed that RF stored at −20 °C yielded gas production results that were comparable to FRF, with no differences in total gas production or lag time over six months, making storage at −20 °C preferable to −80 °C. Additionally, Chaudhry and Mohamed [7] noted that TRF can reliably predict in vitro degradation when FRF is not readily available. This is supported by the findings of Qiu et al. [25] and Pramita et al. [8], who found that freezing at −20 °C or −80 °C did not affect the microbial population composition or fermentation characteristics. Overall, these findings justify the use of TRF in the present study as a reliable alternative to FRF for the fermentation assessment of various feeds.

When compared with RF characteristics reported in previous studies (Table 2), the TRF used in this investigation fell within or near the reported ranges for FRF from the same laboratory [3] and from other laboratories [6], 10], 25]. The pH (7.5) was consistent with prior observations, and ammonia concentrations (127 mg/L) remained within the expected range despite being slightly lower than in the fresh counterpart. Total VFA and major VFAs such as acetate, propionate, and butyrate in TRF also aligned with earlier values, further indicating preserved fermentative potential. The higher isobutyrate level observed in TRF falls within the broader range reported in literature and may reflect microbial adaptation or selective survival of certain species during the freeze–thaw process. These results support the reliability of using TRF as a substitute for FRF in in vitro fermentation trials when immediate access to fresh inoculum is not feasible.

The results showed that various herbs and spices differed significantly in most proximate compositions. These differences may be attributed to variations in plant varieties and climatic conditions in different growing locations [34]. Furthermore, this finding suggests that GTL, GBL, BS, and GB are good sources of protein.

The NDF fraction, which represents the entire cell wall (cellulose + hemicellulose + lignin), ranged from 202 to 391 g/kg DM in herbs, while it ranged from 14 to 548 g/kg DM in spices. These amounts are lower than the aerial fraction reported for several forage sources, which ranged from 595 to 846 g/kg DM [14]. However, our values are comparable to those reported by Ramdani et al. [22] for green tea leaves, Thao et al. [35] for eucalyptus leaves, and Stefenoni et al. [36] for oregano leaves. Notably, OL exhibited the highest NDF content, exceeding the findings of Olijhoek et al. [37], who reported NDF values of 243–289 g/kg DM. However, these values are comparable to those reported by El-Naggar and Ibrahim [38] and Bhargav et al. [39], for CS, as well as by Foiklang et al. [40] and Moate et al. [41], for GrP. In contrast to Al-Naqeep et al. [42], who observed NDF values of 206–271 g/kg DM, black seed exhibited a higher NDF content in this study. Additionally, climatic conditions have an impact; according to Pascual et al. [43], high temperatures and low precipitation are associated with increased cell wall carbohydrates (NDF) and decreased soluble content in various plants.

The ADF (lignocellulose) and ADL (lignin) fractions varied among the investigated herbs. Our results for EL and OL are consistent with previously reported findings [36], 44]. However, EL showed the highest ADF content, surpassing the findings of Thao et al. [35] who reported 220 g ADF/kg DM for EL. Additionally, Kondo et al. [45] determined that the ADF content in green tea by-products was 289 g/kg DM, while Sallam et al. [12] found that EL contained 493–504 g ADF/kg DM, which is higher than the results of the current study. The investigated spices also displayed varying ADF and ADL contents, with results for GrP and CS aligning with those previously reported [39], 41], respectively. While others reported 65–89 g ADF/kg DM [42] and 120 g ADF/kg DM [34] in BS, the current study found a higher ADF proportion. Moreover, Foiklang et al. [40] determined that the ADF content in grape pomace was 306 g/kg DM, and Juráček et al. [46] discovered that dried grape pomace contained 380 g ADF/kg DM, which is lower than the results of the current study. The growth stage is an important factor to consider, and genotype plays a key role in the accumulation of fibres in the cell wall [47].

The GBL was not compared to other findings because, to the best of our knowledge, no data on the proximate analysis of great burnet (S. officinalis L.) were available in the literature.

Preliminary phytochemical profiling provides important information about the variety of distinct groups of secondary metabolites in plant extracts [48]. Plant secondary metabolites have long been considered vital for protecting plants against predators, and their synthesis is influenced by environmental, seasonal, and external stimuli [49]. Although secondary metabolites have traditionally been viewed as hazardous to animals and identified as anti-nutritional agents [50], they have gained popularity in animal nutrition in recent decades due to their positive effects on parasite management, rumen fermentation, and methane reduction [49].

The selected herbs were rich in tannin content. Similar results were reported for TT (77 g GAE/kg), CT (19 g leucocyanidin/kg), and TS (13 g DE/kg) in EL [51]. Furthermore, Singh [52] found the same CT value in OL. Additionally, Samadi and Fatemeh [53] observed comparable TT results in GTL. In contrast, the TT, CT, and TS contents of GTL in this investigation were much lower than those reported previously, which used the same reference standard equivalents 231, 204, 176, and 276 g/kg DM, respectively [22]. Similarly, Sallam et al. [12] noted higher TT levels in EL, whereas Bendifallah et al. [54] found higher amounts in OL, which contrasts with the current study’s findings. On the other hand, the TT and TS levels reported for both OL [55] and EL [56] were lower than the results found in this study.

The selected spices were rich in saponin content. Similarly, Akeem et al. [19] reported comparable CT and TS values in GB, whereas the CT and TS contents of GrP in the current study were much lower than those previously reported by Spanghero et al. [57]. Similarly, Milutinović et al. [58] noted higher amounts of CT in grape pomace. In addition, Khan and Chaudhry [59] discovered higher levels of CT and TS in CS. However, different plants may have various constituents and nutritional benefits depending on where they are cultivated or sourced [42]. Moreover, it was indicated that several factors could influence the phytochemical composition, including growing locations, maturation stages, processing techniques, and isolation methods [24].

High levels of tannins in the diet (6–12 % DM) may reduce animal production and digestive efficiency [60]. More recently, Gerlach et al. [61] found no influence of tannins on the digestibility of concentrate organic matter at a tannin level of 1 %. However, digestibility decreased when the tannin contents were 3 % (−21 %) and 5 % (−28 %). According to Cabral et al. [62], who studied three levels of tannins in sheep diets, low-, medium-, and high-tannin cultivars exhibited distinctly different DM digestibility. Therefore, Besharati et al. [63] demonstrated in their review article that the ingestion of tannins altered digestibility by affecting the pattern of ruminal fermentation. Although these effects are discussed in two subsections, the repeated conclusion is that increased dietary tannin levels could decrease food digestion by raising nitrogen excretion in the faeces. Nevertheless, depending on their quantity, type, chemical structure, and feed composition, condensed tannins can have either positive or negative effects on ruminants, especially when there is a high percentage of crude protein in the diet [64].

Saponins have been studied for their ability to influence rumen fermentation by reducing protozoal populations, thereby lowering hydrogen (H2) availability and methane (CH4) production [65]. Saponins can form complexes with the lipid membranes of bacteria, increasing their permeability, causing an imbalance, and resulting in the lysis of the microorganisms; most saponins do influence protozoa [66]. Moreover, Wallace et al. [67] proposed that saponins disrupt protozoa by forming complexes with sterols on the protozoan membrane surface, leading to impairment and disintegration. Therefore, determining the phytochemical contents of herbs and spices before adding them to the ration is critical for determining the total amount of additives to be used.

The Ankom DaisyII incubator was developed as a rumen simulation equipment to facilitate the measurement of in vitro degradability. The procedure involves degrading multiple feed samples in bags simultaneously within glass jars that are rotated in an enclosed chamber. Compared to traditional techniques, such as the first stage of the Tilley and Terry [68] method and the Van Soest et al. [69] method, the Ankom DaisyII incubator offers advantages in terms of time, efficiency, and labour requirements [70]. Its design allows for the examination of a high number of samples [71], 72]. It is a simple, affordable, and effective instrument for estimating the degradability of a variety of diets and feedstuffs [73] simultaneously. Simply, it can mimic the rumen of four ruminant animals, e.g. sheep, goat, cattle, while placed together in the same anaerobic chamber.

Due to the initial focus on the in vitro degradation of each herb and spice individually, it is challenging to compare the current findings with those of previous investigations where spices and herbs were tested as additives or supplements alongside other feeds. However, the lower concentration of fibre fractions may explain the higher in vitro degradability of GB and OF (Table 5). Conversely, GrP exhibited reduced in vitro degradation of DM and OM contents due to its higher fibre contents (Table 3). This is supported by the negative association between fibre content and the IVDMD and IVOMD values (Table 6).

Due to the complexity of interactions among fibre components and phytochemical contents, multivariate statistical methods were employed to determine key drivers of degradability. Principal Component Analysis (PCA) revealed clustering patterns linked to both fibre content and secondary metabolites, highlighting differentiation between herbs and spices. Additionally, multiple linear regression models were constructed to predict IVDMD using quantitative predictors such as NDF, ADF, and total tannins. The inclusion of categorical variables (e.g., herb vs. spice) helped capture compositional variation, offering practical insights into the degradability potential of individual additives. The two main PCs were the fibre fractions (NDF, ADF, and ADL) and the phytochemical contents (TT, CT, and TS), while the third component was cellulose. These two PCs can be utilised to select herbs and spices as feed additives. However, it is not advisable to rely on a single feature for herb/spice selection, as this may disrupt other related traits; therefore, such issues can be mitigated by calculating and applying PCs. The primary challenges for evaluating herbs and spices in ruminant feeds, whether in vitro or in vivo, are the required labour and high costs. Nevertheless, depending on the selection goal, reducing the number of necessary measurements may make this approach feasible, allowing the final selection to be based on fewer traits. Although labour and cost may continue to be significant issues, such practices should be implemented before utilising herbs and spices in feed. Consequently, it may be possible to rely on one of the first principal components (fibre fractions) when selecting herbs and spices as feed additives, rather than assessing all contents, due to the high correlation between them. The same approach applies to the phytochemical contents as the second principal component.

The multiple regression analysis was performed to assess the impact of phytochemical components (e.g., tannins and saponins) and fibre fractions (e.g., NDF and ADF) on IVDMD across different herbs and spices. Since each plant material possesses unique chemical compositions that may affect ruminal degradation differently, including individual herbs and spices as categorical variables, allowed for a more comprehensive evaluation of their effects.

This approach provided key insights, including the identification of primary degradability drivers, the relative ranking of different feed additives, and potential interaction effects between fibre fractions and secondary metabolites. While correlation analysis identifies pairwise relationships, multiple regression accounts for the simultaneous influence of multiple factors, ensuring that the observed effects are not confounded by interdependencies.

The results indicated that tannins and saponins had no impact on IVDMD across different herbs and spices. However, some individual plant materials (e.g., garlic bulb and onion flesh) exhibited higher IVDMD, whereas others (e.g., grape peel and black seed) showed reduced degradability. These findings suggest that phytochemical composition alone may not be the primary determinant of IVDMD, reinforcing the importance of evaluating both fibre fractions and secondary metabolites together.

Due to the absence of ADL in the fibre fraction assessment, garlic bulbs were not included in the multiple regression analysis. The in vitro dry matter degradability values of OF and GTL showed significant improvement, as indicated by their positive coefficients. This suggests that the lower NDF, ADF, and ADL contents in OF and GTL may contribute to their higher IVDMD. Conversely, the negative coefficients for NDF, ADF, and ADL contents in GrP, EL, BS, and OL indicate a decrease in their IVDMD. This suggests that the fibre and phytochemical contents of each herb and spice indicate their potential as feed additives, given their impact on degradability, which may affect the substrate feed components. The differences in coefficients among the studied herbs and spices highlight various factors influencing IVDMD across different feed groups. These findings contribute to the growing body of knowledge regarding the quality of herbs and spices and guide future research efforts aimed at enhancing nutritional management strategies for optimal ruminant nutrition.

5 Conclusions

This study confirms that TRF can be used effectively as an alternative to FRF for screening and ranking herbs and spices for in vitro degradability, as TRF preserved sufficient microbial activity, as indicated by the fermentation characteristics. The presence of significant quantities of TT and CT in GTL, GBL, and EL, as well as high levels of TS in GBL and all spices, particularly GB and OF, suggests that these herbs and spices might serve as natural additives in ruminant diets. Based on standard evaluations of proximate composition, phytochemical contents, and in vitro degradability, all herbs and spices except GrP appear useful for promoting rumen degradability. However, advanced multivariate analyses (PCA and multiple regression) provided a more precise assessment of feed additive potential, identifying GB, OF, GTL, GBL, CS, and OL, in descending order of effectiveness, as the most promising candidates. Conversely, GrP, EL, and BS may have had minimal or negative impact on degradability. These findings highlight the limitations of relying solely on traditional compositional analysis for feed additive selection and demonstrate that multivariate statistical approaches offer a more meaningful strategy for pre-screening botanicals in ruminant nutrition research. Further studies should explore dose-response effects and validate these findings in vivo before implementation.

Corresponding author: Kawa Merkhan, School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK; and College of Agricultural Engineering Sciences, Duhok University, Duhok/Kurdistan Region, Iraq, E-mail:

  1. Funding information: This study was supported by the Higher Committee for Education Development in Iraq (HCED-IRAQ).

  2. Author contributions: All authors have reviewed and approved the final version of the manuscript for publication. KM conceived, designed, and conducted the study, including data evaluation and analysis. ASC conceived and contributed to the experimental design. KM drafted the original manuscript, which was revised following discussions on data interpretation with ASC and JS. Additionally, ASC provided supervision and participated in the review and editing of the manuscript.

  3. Conflict of interest: The authors declare no competing interests.

  4. Ethical approval: The studies were approved by the Animal Welfare and Ethical Review Board (AWERB) of Newcastle University, UK (Project ID No: 1089) for the use of animal-derived materials. No live animals were involved in this study. Instead, slaughtered steers at an approved abattoir were used as donors to obtain rumen fluid.

  5. Data availability statement: None of the data or models has been submitted to an official repository. The corresponding author can provide the datasets generated and analysed during the current investigation upon reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/opag-2025-0476).

Received: 2025-08-01
Accepted: 2025-10-10
Published Online: 2025-11-04

© 2025 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/opag-2025-0476
Keywords for this article
thawed rumen fluid; fibre; tannin; saponin; degradability; covariate
Creative Commons
BY 4.0

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