Effects of Oregano on Lactation Performance, Nutrient Digestibility, Ruminal Fermentation Parameters, and Methane Emissions in Dairy Cows: A Meta-Analysis

Abstract

Growing concerns regarding antibiotic use in livestock, due to antibiotic resistance and potential human transmission, have led to increased interest in herbs and their derivatives, including essential oils, which possess antimicrobial properties that may enhance overall productivity and serve as a strategy for methane mitigation. The objective of this meta-analysis was to investigate the effects of adding oregano to the diet in different forms (essential oils, plant materials, or leaves) on the dry matter intake (DMI), milk yield (MY), milk components, nutrient digestibility, ruminal fermentation parameters, and methane (CH₄) emissions of dairy cows. A literature search was conducted to identify papers published from 2000 to 2023. Effect size for all outcomes was reported as a standardized means difference (SMD) and raw means difference with 95% confidence intervals. Heterogeneity was determined using the Q test and I² statistic. The results of the meta-analysis indicated that adding oregano had no effect on DMI (SMD = 0.081; p = 0.507) and MY (SMD = 0.060; p = 0.665). Milk fat percentage, milk protein percentage, and milk lactose percentage were not affected by oregano. The addition of oregano to the diet significantly decreased dry matter digestibility (SMD = −0.502; p = 0.013), crude protein digestibility (SMD = −0.374; p = 0.040), and neutral detergent fiber digestibility (SMD = −0.505; p = 0.014). Ruminal pH (SMD = −0.122; p = 0.411), total volatile fatty acids concentration (SMD = −0.038; p = 0.798), acetate (SMD = −0.046; p = 0.757), propionate (SMD = 0.007; p = 0.960), and butyrate (SMD = 0.037; p = 0.801) proportion were not affected by oregano. The addition of oregano to the diet tended to decrease CH₄/DMI (SMD = −0.275; p = 0.095) but did not affect CH₄ production (SMD = −0.156; p = 0.282). Heterogeneity (Q and I²) was non-significant for all parameters. We conclude that the inclusion of oregano in various forms (essential oils, plant materials, or leaves) in the diet of dairy cows reduces nutrient digestibility but does not significantly affect DMI, MY, milk components, ruminal fermentation parameters, or CH₄ production. Future research should focus on optimizing the dosage of oregano (both EOs and plant materials) and exploring the impact of its form on lactation, nutrient digestibility, ruminal fermentation, and CH₄ emissions in dairy cows.

1. Introduction

The past few years have witnessed increasing concerns over the application of antibiotics in livestock production, due to their potential contribution to the emergence of antibiotic-resistant bacteria and the fact that these may be transmitted from animals to humans. Increasing efforts are being made to find replacements in animal nutrition and this has drawn attention toward herbs and their derivatives, including essential oils (EOs) with antimicrobial properties that can modulate rumen fermentation [1,2]. Oregano is an aromatic and medicinal herb from the Origanum genus, part of the Lamiaceae family, primarily found in the Mediterranean region [3,4]. Known for its essential oil, oregano contains two primary constituents, carvacrol and thymol [2,3,4]. Thymol, a type of monoterpene, demonstrates potent antimicrobial properties against various Gram-positive and Gram-negative bacteria. Similarly, carvacrol, a phenolic compound closely related to thymol, also shows strong antimicrobial effects [2,5]. Reports indicate that supplementing oregano EO can modify ruminal fermentation, altering volatile fatty acid (VFA) concentrations and reducing methane (CH₄) emissions by extensively altering the ruminal bacterial community [5].

However, results from in vitro experiments must be interpreted carefully due to a number of limitations including short-term incubation, buffered medium, and inability to replicate the diversity and viability of the microbial population of the rumen. Therefore, the effects observed in vitro may not be fully reliable for in vivo applications [6].

However, experiments on the in vivo effectiveness of oregano for dairy cows have produced various results. Previous studies show that oregano EO supplementation at doses of 50 mg/kg of DM [6] and 0.2 to 1.0 g/kg of DM [7] had no significant impact on dry matter intake (DMI), milk yield (MY), or enteric CH₄ emissions in dairy cows, while Hristov et al. [8] reported a linear decrease in DMI and CH₄ emissions (g/day and g/kg of DMI) and no significant change in rumen fermentation parameters when supplementing the diet of dairy cows with oregano leaves (250, 500, and 750 g/cow per day). The findings published by Olijhoek et al. [9] indicate that substituting grass/clover with oregano plant materials (leaves, stalks, and flowers) at different levels can impact nutrient digestibility in linear, quadratic, and cubic ways, with no effect on DMI, MY, rumen fermentation, and enteric CH₄ emission in dairy cows.

A comprehensive view is needed to assess the results of these experiments to determine the overall effect of oregano on lactation performance, nutrient digestibility, ruminal fermentation, and CH₄ emissions in dairy cows. This comprehensive view can affect lactation performance and ultimately the profitability of dairy farms while also supporting environmental sustainability regarding greenhouse gas emissions.

Meta-analysis is a statistical technique utilized to integrate and summarize treatment effects from different studies, and to analyze factors that may contribute to any observed heterogeneity in outcomes [10]. This meta-analysis is the first to thoroughly investigate these effects, addressing a gap in the existing literature and enhancing our knowledge of oregano’s significance for dairy cows. Therefore, the present study aims to conduct a comprehensive meta-analysis on the effects of adding oregano to the diet on DMI, MY, milk components, nutrient digestibility, ruminal fermentation parameters, and CH₄ emissions in dairy cows.

2. Materials and Methods

2.1. Literature Search

A thorough search of English-language publications from 2000 to 2023 was conducted to find studies investigating the impact of oregano on DMI, MY, milk components, nutrient digestibility, rumen fermentation, and CH₄ emissions in dairy cows. The literature search included three search engines: ISI Web of Knowledge (http://wokinfo.com, accessed on 9 March 2024), Google Scholar (http://scholar.google.com, accessed on 9 March 2024), and PubMed (http://www.ncbi.nlm.nih.gov/pubmed, accessed on 9 March 2024). The keywords used to identify relevant studies included oregano, Origanum vulgare, and dairy cow. The reference lists of all collected articles were examined to ensure that no relevant studies were missed. Full texts were obtained via electronic databases, interlibrary loans, or by contacting the authors directly.

2.2. Inclusion and Exclusion Criteria

Figure A1 presents a PRISMA flow diagram [11] illustrating the data collection process for the meta-analysis. Following the initial search and screening, 16 articles were evaluated for eligibility, with 8 being excluded for the following reasons: mixing oregano EO with other EOs (n = 2), repeating the results of the articles in other articles by the same authors (n = 2), lack of DMI, MY, and milk component reports (n = 2), and failure to report statistical indicators (n = 2). As a result, the 8 articles selected for this meta-analysis met the main criterion, which was the effect of oregano on DMI, lactation performance, nutrient digestibility, rumen fermentation, and CH₄ emissions in dairy cows. However, as Olijhoek et al. [9] reported two experiments, nine experiments were used in total. Two reviewers evaluated all articles according to the inclusion and exclusion criteria. Disagreements were resolved by consulting a third reviewer. A list of the experiments included in the meta-analysis is depicted in

Table 1. Summary of studies included in the meta-analysis on the effects of oregano on lactation performance, nutrient digestibility, ruminal fermentation parameters, and methane emissions in dairy cows.

Reference	CC a	Breed	Forages	Form of Oregano b	Amount of Oregano	Oregano Delivery c	CH4 Measurement Method d
Benchaar, 2020 [6]	1	-	alfalfa silage and corn silage	EO	50 mg/kg of DM	TMR	RC
Hristov et al., 2013 [8]	3	Holstein	corn silage, alfalfa haylage, and grass hay	L	250 g/d, 500 g/d, 750 g/d	TMR	SF6
Kolling et al., 2018 [12]	1	Holstein and Holstein–Gir	corn silage and Tifton hay	EO	0.056% of DM	TP	RC
Lejonklev et al., 2016 [7]	2	Danish Holstein	maize silage and grass–clover silage	EO	0.2 g/kg of DM, 1.0 g/kg of DM	TMR	RC
Olijhoek et al., 2019 [9] (Exp. 1)	3	Danish Holstein	grass/clover silage and maize silage	PM	17.8 g/kg DM, 35.5 g/kg DM, 53.3 g/kg DM	TMR	RC
Olijhoek et al., 2019 [9] (Exp. 2)	3	Danish Holstein	grass/clover silage and maize silage	PM	7 g/kg DM, 13.9 g/kg DM, 20.9 g/kg DM	TMR	RC
Stefenoni et al., 2021 [13]	1	Holstein	corn silage, alfalfa haylage, and grass hay	L	1.77% of DM	TMR	SF6
Stivanin et al., 2019 [14]	1	Jersey	corn silage and pasture	EO	10 g/d	TP	-
Tekippe et al., 2011 [15]	1	Holstein	corn silage, alfalfa silage, and grass/straw hay	L	500 g/d	TP	SF6

^a–d CC, count of comparisons (number of comparisons between the mean treatment group and the mean control group); EO, essential oil; L, leaves; PM, plant material (leaves, stalks, and flowers); TMR, total mixed ration; TP, top-dressed; RC, respiratory chamber; SF₆, sulfur hexafluoride.

.

2.3. Data Extraction

Data extracted from each study included the authors’ names, year of publication, DMI, MY, milk fat percentage (MFP), milk protein percentage (MPP), milk lactose percentage (MLP), ruminal pH, total VFA concentration, proportions of acetate, propionate, and butyrate, dry matter digestibility (DMD), crude protein digestibility (CPD), neutral detergent fiber digestibility (NDFD), daily CH₄ production, and CH₄ production per DMI. All nutrient digestibility values were based on total-tract apparent digestibility. Data such as the number of animals in the control and treatment groups, along with the standard error (SE), were also extracted. The standard deviation (SD) was recorded as the measure of variance. If the SD was not provided in the studies, it was calculated by multiplying the reported SE of the means by the square root of the sample size. The data were entered into Excel spreadsheets (Microsoft Corp., Redmond, WA, USA) and reviewed by two investigators to confirm that the information was accurately transcribed from the papers before conducting statistical analyses. The variables evaluated in this meta-analysis are shown in

Table A1. Variables evaluated in each study for meta-analysis.

Reference	Evaluated Variables a
Benchaar, 2020 [6]	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Hristov et al., 2013 [8]	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Kolling et al., 2018 [12]	DMI, MY, MFP, MLP, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Lejonklev et al., 2016 [7]	DMI, MY, MFP, MPP, CH4, CH4/DMI
Olijhoek et al., 2019 [9] (Exp. 1)	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Olijhoek et al., 2019 [9] (Exp. 2)	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Stefenoni et al., 2021 [13]	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4, CH4/DMI
Stivanin et al., 2019 [14]	DMI, MY
Tekippe et al., 2011 [15]	DMI, MY, MFP, MPP, MLP, DMD, CPD, NDFD, pH, total VFAs, acetate, propionate, butyrate, CH4

^a DMI, dry matter intake; MY, milk yield; MFP, milk fat percentage; MPP, milk protein percentage; MLP, milk lactose percentage; DMD, dry matter digestibility; CPD, crude protein digestibility; NDFD, neutral detergent fiber digestibility; VFAs, volatile fatty acids; CH₄, methane.

.

2.4. Statistical Analysis

Statistical analysis was carried out using Comprehensive Meta-Analysis (CMA) software version 4 (Biostat, Addison, TX, USA) to determine the effect size for DMI, MY, milk components, rumen fermentation, nutrient digestibility, and CH₄ emissions, represented as standardized mean differences (SMD) with a 95% confidence interval (CI). The SMD denotes the mean difference between the treatment and control groups, standardized according to the SD of both groups [16]. The SMD is calculated using the following formula:

$$\mathrm{S}\mathrm{M}\mathrm{D}={\displaystyle \frac{{\overline{\mathrm{x}}}_{\mathrm{e}}-{\overline{\mathrm{x}}}_{\mathrm{c}}}{{\mathrm{S}}_{\mathrm{p}}}}$$

where ${\overline{\mathrm{x}}}_{\mathrm{e}}$ is the experimental group mean, ${\overline{\mathrm{x}}}_{\mathrm{c}}$ is the control group mean, and ${\mathrm{S}}_{\mathrm{p}}$ is the pooled SD [17].

In addition to calculating the SMD, the raw mean difference (RMD) was also determined for each outcome, along with a 95% CI. The RMD represents the difference between the treatment and control groups and allows the effect size to be expressed in the same units as the original measurements. A random effects model was employed for the meta-analysis, based on the assumption that a distribution of effects exists, leading to heterogeneity among the study results [16]. The significance of the effect size estimates (SMD and RMD) was determined at a p-value of ≤0.05. Forest plots were constructed to evaluate the effect of oregano on MY and CH₄/DMI. The effect size for the forest plot was the SMD at a 95% CI using the random effects model.

Statistical heterogeneity indicates that the true effects vary across studies, meaning they are not identical in each one [10]. The presence of heterogeneity indicates underlying differences in the clinical characteristics of the herds, variations in study design, and statistical diversity [17]. Heterogeneity was assessed using the Chi-square (Q) test and the I² statistic [16]. Variations among the study level were assessed using a Q test. The significance level was set at 0.1 due to the relatively low power of the Q test when only a small number of studies are included [17,18]. Although the Q test is useful for detecting heterogeneity, the I² statistic was used to quantify the extent of heterogeneity as a percentage [17].

$$I^2\left(\mathrm{\%}\right)={\displaystyle \frac{Q-\left(\mathrm{k}-1\right)}{Q}}\times 100$$

where Q is the X² heterogeneity statistic and k is the number of trials. The I² statistic describes the percentage of variation across studies attributable to heterogeneity. Negative values of I² are set to zero, so the statistic ranges from 0% to 100% [17]. An I² value between 0% and 40% may indicate negligible heterogeneity, 30% to 60% could suggest moderate heterogeneity, 50% to 90% may indicate significant heterogeneity, and 75% to 100% could reflect a high level of heterogeneity [19].

Although a meta-analysis offers a mathematically precise synthesis of the included studies, if these studies are a biased representation of the relevant research, the calculated mean effect will likewise be biased. This issue, referred to as publication bias, was evaluated using Egger’s linear regression asymmetry test [20]. When significant bias (p < 0.10) was detected, the trim-and-fill method [21] was applied to estimate the number of missing observations. Funnel plots were used to illustrate asymmetry; these plots assume that, in the absence of publication bias, effect sizes would be symmetrically distributed around the true effect size, indicating that extreme results have not been omitted.

3. Results

3.1. Characteristics of the Database

The studies selected for this meta-analysis are presented in Table 1. Olijhoek et al. [9] reported the results of two experiments, resulting in a total of nine experiments examined in this meta-analysis. Four studies utilized oregano EO as a dietary additive for dairy cows [6,7,12,14]. Among these, two studies applied the oregano EO as a top-dressing [12,14], while the other two incorporated it into the total mixed ration [6,7]. Three studies incorporated oregano leaves into the diet [8,13,15], with only Hristov et al. [8] administering oregano leaves as a top-dressing. Olijhoek et al. [9] included oregano plant materials (leaves, stalks, and flowers) as a partial replacement for grass/clover silage in the dairy cow diet. Methane emission measurements were conducted using the sulfur hexafluoride (SF₆) tracer technique in three studies [8,13,15] and the respiratory chamber method in four studies [6,7,9,12]. In the study by Stivanin et al. [14], the experiment focused on cows during the transition period, and the results for DMI and MY were evaluated during the post-calving phase. The breeds of dairy cows included Holstein [8,13,15], Danish Holstein [7,9], Jersey [14], and Holstein–Gir and Holstein [12].

3.2. Dry Matter Intake, Milk Yield, and Milk Components

The results of the meta-analysis on the effect of adding oregano to the diet on DMI, MY, and milk components in dairy cows are reported in

Table 2. Effect size and heterogeneity of oregano supplementation on dry matter intake, milk yield, and milk components in dairy cows.

Outcomes a	CC b	SMD c (95% CI d)		Heterogeneity			RMD e (95% CI)	Publication Bias
		Random Effect	p-Value	Q	p-Value	I2	Random Effect	Egger (p-Value)
DMI, kg/d	23	0.081 (−0.157, 0.318)	0.507	7.978	0.997	0.0	0.274 (−0.084, 0.631)	0.298
MY, kg/d	16	0.060 (−0.211, 0.331)	0.665	6.234	0.976	0.0	0.216 (−0.571, 1.002)	0.746
MFP, %	15	−0.054 (−0.338, 0.231)	0.711	11.441	0.651	0.0	−0.061 (−0.181, 0.058)	0.238
MPP, %	14	−0.021 (−0.314, 0.271)	0.886	1.410	1.00	0.0	0.009 (−0.037, 0.055)	0.139
MLP, %	13	−0.180 (−0.471, 0.110)	0.224	2.505	0.998	0.0	−0.019 (−0.056, 0.017)	0.721

^a–e DMI, dry matter intake; MY, milk yield; MFP, milk fat percentage; MPP, milk protein percentage; MLP, milk lactose percentage; CC, count of comparisons (number of comparisons between the mean treatment group and the mean control group); SMD, standardized mean difference; CI, confidence interval; RMD, raw mean difference.

. Including oregano in the diet had no effect on DMI (SMD = 0.081; p = 0.507) and MY (SMD = 0.060; p = 0.665). Figure 1 illustrates the forest plot, which presents the results from individual studies as well as the overall outcome for MY. Heterogeneity was not significant for DMI and MY (Q and I², Table 2). Milk fat percentage (SMD = −0.054; p = 0.711), MPP (SMD = −0.021; p = 0.886), and MLP (SMD = −0.180; p = 0.224) were not affected by oregano. Heterogeneity was non-significant for milk components (Q and I², Table 2). There was no indication of publication bias for DMI, MY, and milk components (p > 0.10).

3.3. Nutrient Digestibility

The results of the meta-analysis on the effect of adding oregano to the diet on nutrient digestibility in dairy cows are reported in

Table 3. Effect size and heterogeneity of oregano supplementation on nutrient digestibility in dairy cows.

Outcomes a	CC b	SMD c (95% CI d)		Heterogeneity			RMD e (95% CI)	Publication Bias
		Random Effect	p-Value	Q	p-Value	I2	Random Effect	Egger (p-Value)
DMD, %	11	−0.502 (−0.895, −0.108)	0.013	13.022	0.222	23.207	−0.886 (−1.390, −0.382)	<0.1
CPD, %	12	−0.374 (−0.730, −0.017)	0.040	13.561	0.258	18.887	−0.822 (−1.479, −0.166)	<0.1
NDFD, %	12	−0.505 (−0.907, −0.103)	0.014	16.486	0.124	33.276	−1.448 (−2.383, −0.513)	<0.1

^a–e DMD, dry matter digestibility; CPD, crude protein digestibility; NDFD, neutral detergent fiber digestibility; CC, count of comparisons (number of comparisons between the mean treatment group and the mean control group); SMD, standardized mean difference; CI, confidence interval; RMD, raw mean difference.

. The use of oregano in the diet significantly decreased DMD (SMD = −0.502; p = 0.013), CPD (SMD = −0.374; p = 0.040), and NDFD (SMD = −0.505; p = 0.014). Heterogeneity for nutrient digestibility was not found to be significant (Q and I², Table 3). Publication bias was detected for DMD, CPD, and NDFD. The trim-and-fill method indicated three missing observations for DMD, five missing observations for CPD, and four missing observations for NDFD in the funnel plot (Figure A2, Figure A3 and Figure A4).

3.4. Rumen Fermentation Parameters

The results of the meta-analysis on the effect of adding oregano to the diet on rumen fermentation parameters in dairy cows are reported in

Table 4. Effect size and heterogeneity of oregano supplementation on rumen fermentation parameters in dairy cows.

Outcomes a	CC b	SMD c (95% CI d)		Heterogeneity			RMD e (95% CI)	Publication Bias
		Random Effect	p-Value	Q	p-Value	I2	Random Effect	Egger (p-Value)
pH	13	−0.122 (−0.413, 0.169)	0.411	3.879	0.986	0.0	−0.012 (−0.046, 0.023)	0.161
Total VFAs, mM	13	−0.038 (−0.329, 0.253)	0.798	4.615	0.970	0.0	−0.863 (−4.313, 2.587)	0.013
Acetate, mol/100 mol	13	−0.046 (−0.336, 0.244)	0.757	3.129	0.995	0.0	−0.025 (−0.624, 0.574)	0.248
Propionate, mol/100 mol	13	0.007 (−0.248, 0.299)	0.960	5.615	0.934	0.0	−0.057 (−0.457, 0.343)	0.109
Butyrate, mol/100 mol	13	0.037 (−0.254, 0.329)	0.801	5.873	0.922	0.0	0.216 (−0.181, 0.613)	0.123

^a–e VFAs, volatile fatty acids; CC, count of comparisons (number of comparisons between the mean treatment group and the mean control group); SMD, standardized mean difference; CI, confidence interval; RMD, raw mean difference.

. The use of oregano in the diet did not affect ruminal pH (SMD = −0.122; p = 0.411) or total VFA concentration (SMD = −0.038; p = 0.798). The meta-analysis also revealed that the proportions of acetate (SMD = −0.046; p = 0.757), propionate (SMD = 0.007; p = 0.960), and butyrate (SMD = 0.037; p = 0.801) were not significantly different. Heterogeneity was not significant for rumen fermentation parameters (Q and I², Table 4). Publication bias was detected for total VFA concentration (p < 0.10). The trim-and-fill method identified five missing observations for total VFA concentration in the funnel plot (Figure A5).

3.5. Methane Production

The results of the meta-analysis on the effect of adding oregano to the diet on methane production in dairy cows are reported in

Table 5. Effect size and heterogeneity of oregano supplementation on methane emissions in dairy cows.

Outcomes	CC a	SMD b (95% CI c)		Heterogeneity			RMD d (95% CI)	Publication Bias
		Random Effect	p-Value	Q	p-Value	I2	Random Effect	Egger (p-Value)
CH4, g/d	15	−0.156 (−0.441, 0.128)	0.282	11.100	0.678	0.0	−2.221 (−16.606, 12.165)	0.269
CH4/DMI, g/kg	14	−0.275 (−0.599, 0.048)	0.095	14.544	0.337	10.617	−0.340 (−0.915, 0.236)	0.101

^a–d CC, count of comparisons (number of comparisons between the mean treatment group and the mean control group); SMD, standardized mean difference; CI, confidence interval; RMD, raw mean difference.

. The use of oregano tended to decrease CH₄/DMI (SMD = −0.275; p = 0.095; Figure 2) but did not significantly affect daily CH₄ production (SMD = −0.156; p = 0.282). Heterogeneity was not found to be significant for CH₄ production and CH₄/DMI (Q and I², Table 5). No publication bias was found for CH₄ and CH₄/DMI (p > 0.10).

4. Discussion

The results of this meta-analysis indicated that adding oregano to the diets of dairy cows (in the form of EOs, plant materials, or leaves) has no significant impact on DMI. This finding aligns with most of the studies included in the analysis, except for those by Hristov et al. [8] and Stivanin et al. [14]. By supplementing dairy cow diets with different amounts of oregano leaves (0, 250, 500, and 750 g/d, corresponding to approximately 8.1, 16.6, and 25.8 g of oregano DM/kg of dietary DM, respectively), Hristov et al. [8] reported a significant decrease in DMI, with a 5% drop in DMI compared to the control group at the greatest supplementation level compared, probably due to the strong objectionable odor of its EO compounds [22]. In contrast, Olijhoek et al. [9] reported no impact of oregano on DMI both during digesta collection and during gas measurements, and this lack of impact can be attributed to ineffectiveness of oregano on diet palatability. It appears that a number of various factors, including EO content, type and amount of feed (forage and concentrate), and adaptation of the microbial population in the rumen contribute to changing or fixed DMI [23,24].

The findings of the present meta-analysis demonstrate that adding oregano to the diet of dairy cows does not significantly influence MY (RMD = 0.216 kg/d), which is in line with the studies included in the meta-analysis, except for Olijhoek et al. [9] and Stivanin et al. [14]. By including different levels of oregano leaves (17.8, 35.5, and 53.3 g DM/kg of dietary DM) in dairy cow diets, Olijhoek et al. [9] reported a significant quadratic effect on MY, and Stivanin et al. [14] also reported a 15% increase in MY compared to their control group. A meta-analysis by Dorantes-Iturbide et al. [25] reported that EO supplementation in diets for ruminants increases MY, probably due to improved nutrient digestibility, reduced CH₄ emissions, and reduced ruminal NH₃-N concentration. However, our meta-analysis showed no improvement in nutrient digestibility and ruminal fermentation (content of glucogenic VFAs, particularly propionate).

Regarding MFP, Kolling et al. [12] reported that adding oregano EO to dairy cow diets reduces MFP by 9%, while Tekippe et al. [15] reported a 5% increase in the MFP of dairy cows supplemented with oregano leaves compared to the control group, perhaps due to minor changes in ruminal microbial fermentation and an increase in the acetate:propionate ratio. However, the findings of the present meta-analysis indicated no significant reduction in MFP, consistent with the results of the studies contained in this meta-analysis. Since the mammary gland in ruminants is the primary precursor for the de novo synthesis of fatty acids based on ruminal acetate, no increase in ruminal acetate concentration was observed in this meta-analysis, consistent with the meta-analysis conducted by Dorantes-Iturbide et al. [25].

Regarding MPP, Olijhoek et al. [9] used oregano (plant material) at different levels (17.8, 35.5, and 53.3 g DM/kg of dietary DM) for dairy cow diets and reported a significant effect that tended toward a quadratic effect, while the results of our meta-analysis indicated that oregano supplementation has no impact on MPP, consistent with the studies included in the present meta-analysis. Regarding MLP, Stefenoni et al. [13] observed a significant reduction in MLP using oregano leaf supplementation (1.7% DM) for dairy cow diets (4.94% vs. 4.87%), while our meta-analysis results, in line with the other studies included herein, showed that oregano has no significant effect on MLP.

The inclusion of EOs in diets is partly intended to increase the relative frequency of the microbial population of the rumen and improve nutrient usability, as a close connection exists between relative nutrient digestibility and certain ruminal microorganisms [26]. In the present meta-analysis, we were not able to analyze ruminal microorganisms and find how they are connected to nutrient digestibility because most studies included here, except for Hristov et al. [8] and Tekippe et al. [15], did not report populations of bacteria, archaea, and fungi in the rumen. However, the results of the present meta-analysis revealed that adding oregano to the diet of dairy cows reduces the digestibility of DM, CP, and NDF, with a numerically small reduction of about 0.8% for DM and CP, and about 1.4% for NDF. Hristov et al. [8] and Olijhoek et al. [9] reported an influence on NDF digestibility. In the experiments conducted by Olijhoek et al. [9], grass/clover silage was substituted with plant materials (leaves, stalks, and flowers) and, although the NDF was the same for the experimental groups in both experiments, the indigestible neutral detergent fiber (iNDF) content was greater in the diets containing oregano, and this probably contributed to the reduced NDF digestibility. Olijhoek et al. [9] reported an iNDF content of 289 g/kg DM for oregano (leaves, stalks, and flowers) in their first experiment and 112 g/kg DM and 44 g/kg DM for grass/clover silage in their first and second experiments, respectively. In addition, in their second experiment, where the experimental diets contained greater amounts of EOs compared to the first experiment, an influence on CP digestibility was observed, indicating that EOs can influence protein digestibility. Although the in vitro experiment by Zhou et al. [5] demonstrated that the increased inclusion of oregano EO (13, 52, 91, 130 mg/L) gradually increased DM and NDF digestibility, with the greatest digestibility found at the dosage of 52 mg/L, the experiments conducted in vivo did not show improved nutrient digestibility [6,8,9]. It is important to note that many EO concentrations that positively influence ruminal fermentation measured in vitro may have different effects when tested in vivo, as the fermentation processes in these two conditions are not the same. This discrepancy should be acknowledged as a potential factor contributing to variations in the results.

It has been shown that the use of oregano EO under in vitro conditions can alter rumen fermentation, leading to a linear decrease in total VFAs, acetate, propionate, and butyrate with increasing inclusion rates of oregano EO [5].

However, the results of the present meta-analysis for in vivo measurements indicate that adding oregano (in the form of EOs, plant materials, or leaves) to the diet of dairy cows has no effect on ruminal pH, total VFAs, acetate, propionate, and butyrate. This is consistent with the findings of the studies included in our meta-analysis. Although butyrate concentration was not influenced in this meta-analysis, Hristov et al. [8] reported a significant reduction in butyrate concentration in cows that received oregano.

Over the past few years, a large number of studies have been documented on the antimicrobial activities of herbs, indicating that phytochemicals can be used to selectively inhibit methanogens in the rumen [27,28]. Although most studies were conducted in vitro and over a short period of time, the results show that EOs and their active ingredients may favorably change rumen fermentation [1] and influence CH₄ production, as several in vitro studies including Zhou et al. [5] assessed the antimethanogenic properties of oregano. The study by Zhou et al. [29] demonstrated that supplementation with oregano EO can manipulate the rumen microbial population in sheep. The effects were dose dependent, with 4 g/day benefiting the microbial population, while 7 g/day had detrimental effects.

In the present meta-analysis, based on the effect size of RMD, CH₄ emission decreased by about 2.221 g/day; the effect size was not significant but the effect size of SMD on CH₄/DMI tended toward a significant decrease. These findings should be interpreted cautiously given the relatively small number of studies on CH₄ emission included in our meta-analysis and the differences in measurement techniques. Studies conducted in respiration chambers demonstrated that oregano EO [6,7,12] and plant materials of oregano [9] had no impact on enteric CH₄ production and CH₄/DMI (except for the experiment conducted by Kolling et al. [12]). Kolling et al. [12] reported that CH₄/DMI in cows fed oregano extract tended to decrease, probably due to increased DMI during measurement in the respiration chamber, while outside respiration chambers, oregano had no impact on DMI. Reduction in CH₄ (g/kg of DMI) in the cows that received oregano extract in the experiment conducted by Kolling et al. [12] is not supported by the data on rumen fermentation and nutrient digestibility, and therefore reduction in CH₄ production with increased DMI may be related to increased passage rate in the rumen [6]. Olijhoek et al. [9] reported no impact of increased dried whole-plant oregano on enteric CH₄ production, together with no changes in DMI and rumen fermentation.

Within the studies selected for the present meta-analysis, three studies [8,13,15] assessed CH₄ emission using the SF₆ technique. In Tekippe et al. [15], feed supplementation using 500 g of oregano leaves led to a 40% reduction in CH₄ production but had no impact on DMI. In Hristov et al. [8], the cows fed different levels of oregano leaves (250, 500, and 700 g/day) tended to reduce CH₄ production (g/d) and increase CH₄/DMI, while Stefenoni et al. [13] found no significant changes in CH₄ production (g/d) and CH₄/DMI. In experiments assessing CH₄ production using the SF₆ technique, reduction in CH₄ production was not consistent with changes in rumen fermentation, nutrient digestibility, protozoa count, and Methanobrevibacter. Generally, although herbs and their EOs are expected to change fermentation pathways and eventually reduce CH₄ by modifying rumen fermentation properties, improving DM and organic matter degradation, enhancing fiber digestibility, and thereby reducing the acetate:propionate ratio [5,28], in the present meta-analysis, as well as the studies included herein, no such changes were observed. It is also important to note that the observed effects on CH₄ emission in a number of studies cannot be attributed to EOs or their major ingredients like carvacrol. In other words, some compounds other than EOs, such as lipids, FAs, phenolics, quinones, and flavonoids, may also inhibit rumen methanogens [6].

5. Conclusions

The results from this meta-analysis indicate that adding oregano in various forms (EOs, plant materials, or leaves) to dairy cow diets has no effect on DMI, MY, and milk components. While the findings suggest a reduction in DM, CP, and NDF digestibility, no significant changes were observed in rumen fermentation parameters or CH₄ emission. These results are important for environmental sustainability, as they suggest that oregano supplementation may not significantly reduce methane emissions in dairy cows. Future research should focus on optimizing the dosage of oregano (for both EOs and plant materials), exploring the impact of the oregano form, and investigating different feed diets, particularly with regard to forage type. Additionally, studies should investigate how these factors influence the microbial population in the rumen and their relationship to CH₄ emission reduction.