Essential Oils as a Dietary Additive for Laying Hens: Performance, Egg Quality, Antioxidant Status, and Intestinal Morphology: A Meta-Analysis

Abstract

This meta-analysis aimed to evaluate the effects of dietary supplementation with essential oils (EOs) on egg production and quality, antioxidant status in blood serum, and the intestinal morphology of laying hens. The data used were obtained from 38 peer-reviewed publications. The effect size was evaluated by weighted mean differences (WMD) between the experimental treatments (diets added with EOs) and the control treatments (diets without EOs). EO supplementation increased (p < 0.001) egg production (WMD = 2.171%), egg weight (WMD = 0.636 g), egg mass (WMD = 1.679 g/d), and decreased the feed conversion ratio (WMD = −0.074 g/g; p < 0.001). In addition, greater (p < 0.05) eggshell thickness (WMD = 14.262 mm), eggshell strength (0.080 kg/cm²), albumen height (WMD = 0.201 mm), Haugh unit (WMD = 1.102), and yolk color (WMD = 0.071) were observed in response to EO supplementation. In blood serum, the dietary inclusion of EOs increased (p < 0.05) the levels of superoxide dismutase (WMD = 1.147 U/mL), glutathione peroxidase (WMD = 879.553 U/mL), and total antioxidant capacity (WMD = 1.163 U/mL). In the duodenum, jejunum, and ileum, a higher (p < 0.05) villus height (VH), crypt depth (CD), villus width, and VH/CD ratio was observed in response to EO supplementation. In conclusion, the dietary inclusion of essential oils can be used as a nutritional strategy to improve egg production and quality, the antioxidant status of blood serum, and intestinal morphology in laying hens.

1. Introduction

Eggs from laying hens are a high-quality food because they contain nutrients such as proteins, vitamins, minerals, and lipids, which are important in human nutrition [1]. Currently, the demand for eggs from laying hens has increased due to the increase in the human population worldwide [2]. However, laying hens in commercial environments are frequently exposed to a wide variety of stressors such as nutritional (presence of oxidized fats or mycotoxins in the feed), environmental (i.e., heat or cold stress), and physiological (e.g., high rate of egg production and aging) [3,4]. According to Puppel et al. [5], each of these factors contributes to the increase in the production of reactive oxygen species (ROS) in the animal, which can result in alterations in the redox balance and lead to oxidative stress (OS) in birds [4,6]. In laying hens, OS can negatively affect productive performance, egg quality, and bird health [3,7]. Some authors [4,8] have mentioned that including natural antioxidants in the diets of laying hens and broilers can help reduce ROS overproduction and result in lower OS.

According to Nehme et al. [9], EOs are currently among the most economically relevant natural products with antioxidant properties. EOs are volatile and aromatic oily liquids derived from plants, which are obtained by distillation and contain complex mixtures of low molecular weight molecules, mainly terpenes, terpenoids, and phenylpropanoids [10,11]. Although EOs have been used mainly in human pharmacology and cosmetology, their use in domestic animal feed has increased in recent years [12,13]. Beef cattle, dairy cows, and small ruminants are some animals in which EOs are being evaluated more frequently, mainly as nutritional strategies to manipulate rumen fermentation and reduce environmental impact [11,13,14]. However, some recently published review articles [8,15,16,17] suggest that the inclusion of EOs in diets for poultry can be used successfully to improve the oxidative status in blood, the health of the intestinal epithelium, and the quality of derived products (meat and eggs).

Particularly in laying hens, some studies have evaluated the effects of EOs as a dietary additive on productive performance [18,19], egg quality [20,21], antioxidant status in blood serum [10,22], and intestinal morphology [23,24]. However, the observed results have been inconsistent and contradictory in several of these studies. For example, some studies have reported up to 7% higher egg production, 15% better feed conversion, 81% higher total blood antioxidant capacity, and 24% longer intestinal villi in laying hens supplemented with EOs [10,18,19]. However, in other studies [22,23,24] using laying hens, less than 2% of benefits in egg production, feed conversion, and intestinal villi have been detected in response to EO supplementation. These results could be attributed to the high variability between studies regarding age and breed/strain of laying hens, supplementation periods, doses, and primary bioactive metabolites of the EOs used [8,15]. It has been reported that this type of variability can be overcome by using meta-analytic methods [25]. Meta-analysis is a statistical method with high analytical power [26] that uses rigorous procedures that allow one to statistically combine and analyze datasets from various experiments to obtain objective evidence on the effect of a treatment [27,28].

The hypothesis of the present meta-analysis establishes that the inclusion of EOs in the diets of laying hens will benefit egg production and quality, antioxidant status in blood serum, and intestinal morphology. Therefore, this study aimed to evaluate, through a meta-analytic approach, the effects of dietary supplementation with essential oils on egg production and quality, antioxidant status in blood serum, and the intestinal morphology of laying hens.

2. Materials and Methods

2.1. Literature Search

A systematic and exhaustive search of the literature published in English was carried out to identify experiments that evaluated the effects of including EOs in diets for laying hens. The bibliographic search was carried out in the Scopus, ScienceDirect, PubMed, and Web of Science databases and was restricted to documents published between January 2012 and April 2023. The keywords used in the searches were the following: essential oils, laying hens, egg production, egg quality, antioxidant status, and intestinal morphology. The PRISMA guidelines [29] were used in the identification, selection, choice, and inclusion of all documents, as shown in Figure 1.

2.2. Inclusion Criteria

Initial searches returned 315 potential documents, which were reduced to 254 after removing duplicate documents. Subsequently, the rest of the documents were reviewed in detail, and only those that met the following inclusion criteria were included in the final database [27,28,30]: (1) scientific articles published in peer-reviewed journals and written in English; (2) studies conducted with laying hens under confined conditions (i.e., studies under grazing conditions were not considered); (3) studies reporting data on egg production, egg quality, blood serum antioxidant status, or intestinal morphology; (4) studies that used similar diets for the control and experimental treatments, except for the inclusion of EOs in the diets; (5) studies that indicated the doses of EOs (mg/kg DM) added to the experimental diets or provided sufficient information to estimate them; (6) studies that reported the means of the experimental treatments (diets added with EOs) and control (diets without EOs), the number of experimental units (n), and the standard error (SEM) or standard deviation of treatment means.

2.3. Data Extraction

After applying the inclusion criteria, only 38 peer-reviewed scientific articles were selected (

Table 1. Description of the studies included in the meta-analysis database.

Reference	Hen’s Age, Weeks	Breed/Strain	Supplementation Period, Days	Dose (mg/kg Feed)	Primary Bioactive Compound
Abdel-Wareth and Lohakare [18]	32–38	Bovans Brown	42–84	74–295	Menthol
Abo Ghanima et al. [19]	28–76	ISA Brown	56–336	300	Blend
Abo Ghanima et al. [20]	28–76	ISA Brown	56–336	300	Thymol, carvacrol, eugenol
Akbari et al. [32]	42	Lohmann White	56	100–200	Menthol, thymol, blend
Arslan et al. [23]	48	Brown Nick H&N	84	50–200	Blend
Bozkurt et al. [21]	36–43	Lohmann White	56–112	24	Blend
Bozkurt et al. [33]	52	Lohmann White	70	24	Blend
Bozkurt et al. [34]	22	Lohmann Brown	252	24	Blend
Bozkurt et al. [35]	82	White Leghorn	175	24	Carvacrol
Beyzi et al. [36]	120	White Leghorn	70	300	Carvacrol
Cheng et al. [10]	65	Lohmann White	56	75–150	Blend
Cufadar et al. [37]	40	Super Nick H&N	84	50–250	Blend
Ding et al. [38]	54–62	Lohmann White	28–84	50–150	Blend
Feng et al. [24]	60–69	Hy-Line Brown	28–84	5–20	Blend
Gao et al. [39]	58–62	Not reported	28–56	8–24	Blend
Ghanem et al. [40]	24	Lohmann Brown	90	50–150	Cinnamaldehyde
Gul et al. [41]	22	Lohmann White	56	200–600	Blend
He et al. [42]	30	Hy-Line White	49	50–150	Carvacrol
Kavan et al. [43]	60	Hy-Line White	56	100–200	Blend
Kaya et al. [44]	36	Lohmann White	56	150–300	Carvacrol
Laptev et al. [45]	52	Lohmann White	27	90	Blend
Marume et al. [46]	18	White Leghorn	56	1000, 2000	Blend
Migliorini et al. [47]	59–67	Not reported	28–84	50–200	Blend
Mousavi et al. [48]	40–45	Hy-Line White	35–70	100–200	Menthol
Mousavi et al. [49]	40–45	Hy-Line White	35–70	100–200	Menthol
Olgun [50]	21	Super Nick H&N	84	25–600	Blend
Puvaca et al. [51]	55–59	Lohmann Brown	28–56	40–80	Terpinen-4-ol
Ramírez et al. [52]	70	ISA Brown	56	80–150	Thymol
Reshadi et al. [53]	66	Lohmann White	84	250	Pulegone
Rodjan et al. [54]	36	Hisex Brown	28	9.6–77.3	Diallyl trisulfide
Rodjan et al. [2]	36	Hisex Brown	28	9.6–77.3	Diallyl trisulfide
Saleh et al. [55]	24	Bovans Brown	42	250–500	Blend
Torki et al. [56]	42	Lohmann White	56	40	Blend
Torki et al. [57]	30–41	Lohmann White	42–84	150–200	Alpha-pinene, carvonene
Torki et al. [58]	42–52	Lohmann White	28–84	250–500	Linalool, limonene, blend
Wang et al. [59]	52–55	Hy-Line Brown	21–42	100	Blend
Xiao et al. [1]	55	Golden Phoenix	56	300	Blend
Yu et al. [22]	26	Hy-Line Brown	28–56	200–600	Trans-anethole

). First, from each of the selected articles, the following information was extracted: (1) author and year of publication; (2) age of laying hens (in weeks); (3) breed/strain of laying hens; (4) supplementation period (days); (5) doses of EOs added to the experimental diets (mg/kg DM); (6) the primary bioactive metabolite of the EOs. Subsequently, the following response variables were collected from the selected scientific articles: average daily feed intake (ADFI), egg production (EP), egg weight (EW), egg mass (EM), feed conversion ratio (FCR), eggshell thickness (ET), eggshell strength (ES), albumen height (AH), Haugh unit (HU), yolk color (YC), yolk index (YI), malondialdehyde (MDA) in the yolk, enzyme antioxidants in blood serum (superoxide dismutase (SOD), glutathione peroxidase (GPx), total antioxidant capacity (TAC) and MDA), and intestinal morphology (villus height (VH), crypt depth (CD), villus width (VW), and VH/CD ratio) in the duodenum, jejunum, and ileum. The following data were obtained from each of these response variables: (1) means of control treatments (diets without EOs) and experimental treatments (diets added with EOs); (2) n; (3) SEM or SD. When the SD was reported, the values were used directly; however, when the publications did not report a SD, its value was estimated using the SEM value and n through the equation proposed by Higgins and Thomas [31]: $SD=SEM\times \sqrt{n}$. Finally, only the response variables reported in at least three different publications were analyzed since other authors [27,28,30] have mentioned that this allows one to obtain statistically robust results.

2.4. Calculations and Statistical Analysis

In this study, the metafor package [26] of the statistical software R version 4.1.2 was used to perform all of the statistical analyses. The effects of EO supplementation on egg production and quality, antioxidant status in blood serum, and the intestinal morphology of laying hens were evaluated by examining the weighted mean differences (WMD) between the experimental treatments (diets supplemented with EOs) and control treatments (diets without EOs). Treatment means were weighted by the inverse of the variance following the method proposed by Der-Simonian and Laird [60] for a random effects model.

2.5. Heterogeneity and Publication Bias

The consistency of the results between studies was assessed with the chi-square (Q) test, in which, due to its relatively low power, a significance level of p ≤ 0.10 was used [61]. As a complement, the ${\mathrm{I}}^2$ statistic was used to quantify the proportion of variation due to heterogeneity [62]. Values of ${\mathrm{I}}^2$ < 25% indicate low heterogeneity, ${\mathrm{I}}^2$ between 25 and 50% indicates moderate heterogeneity, and I² > 50% indicates high heterogeneity [62]. Finally, Egger’s regression asymmetry test [63] and Begg’s adjusted rank correlation [64] were used to assess the presence of publication bias. Both tests were considered significant when p ≤ 0.05.

2.6. Meta-Regression and Subgroup Analysis

Sources of heterogeneity in the response variables were assessed with meta-regression analysis using the method of moments of Der-Simonian and Lair [60] as this method is well-established for estimating between-study variance. The criteria considered to be able to apply the meta-regression analysis to a response variable were the following: (1) response variable reported in ten or more different scientific articles [65]; (2) p-value ≤ 0.10 for the Q test or ${\mathrm{I}}^2$ > 50% [61]; (3) p-value > 0.05 for the Egger [63] and Begg [64] tests. The age of the laying hens (in weeks), the period of supplementation (in days), and the dose of EOs (mg/kg DM) added to the experimental diets were used as continuous covariates. Likewise, the breed/strain of the laying hens and the primary bioactive metabolite of the EOs were used as categorical covariates. When meta-regression was statistically significant (p ≤ 0.05) for continuous covariates, WMD was assessed with subgroup analyses as follows: age of laying hens (≤45 and >45 weeks), period of supplementation (≤56 and >56 days), and EOS dose (≤150 and >150 mg/kg DM). On the other hand, the WMD of the categorical covariates that were significant (p ≤ 0.05) in the meta-regression were analyzed with the following subgroups: breed/strain of laying hens (Hisex Brown, Hy-Line White, Hy-Line Line Brown, Super Nick H&N, White Leghorn, Lohmann Brown, Lohmann White, ISA Brown, and Bovans Brown) and primary bioactive metabolite of EOs (trans-anethole, limonene, linalool, carvonene, alpha-pinene, diallyl trisulfide, terpinen-4-ol, cinnamaldehyde, carvacrol, thymol, menthol, and blend).

3. Results

3.1. Study Attributes

In the database of the present meta-analysis, the age of the laying hens used in the different studies ranged from 22 to 120 weeks. The main breeds/strains of laying hens used in the studies were Lohman White (31.6%), Hy-Line White (10.5%), ISA Brown (7.9%), Lohman Brown (7.9%), and Hy-Line Brown (7.9%), while the remaining studies used another five different breeds/strains of laying hens. Likewise, the supplementation periods with EOs ranged between 27 and 336 days. At the same time, the doses of EOs used in the present meta-analysis ranged between 24 and 2000 mg/kg DM. The EOs with mixtures of primary bioactive compounds were the most used (52.6%) among the different studies. In addition, a significant proportion of the studies used EOs with menthol (10.5%), thymol (7.9%), and carvacrol (13.1%) as the primary bioactive compound while in the remaining studies (15.9%), the EOs used contained another ten different primary bioactive compounds.

3.2. Performance

EOs supplementation did not affect the ADFI (p > 0.05;

Table 2. Egg performance of laying hens supplemented with essential oils.

Item	N (NC)				Heterogeneity		Egger Test 1	Begg Test 2
		Control Means (SD)	WMD (95% CI)	p-Value	p-Value	I2 (%)	p-Value	p-Value
ADFI, g/d	31 (132)	2.235 (0.87)	−0.047 (−0.410; 0.316)	0.798	<0.001	89.69	0.110	0.062
Egg production (EP), %	31 (132)	83.82 (8.08)	2.171 (1.570; 2.772)	<0.001	<0.001	86.20	0.281	0.478
Egg weight (EW), g/d	30 (115)	59.48 (4.35)	0.636 (0.470; 0.802)	<0.001	<0.001	77.69	0.086	0.169
Egg mass (EM), g/d	23 (95)	50.28 (7.38)	1.679 (1.118; 2.240)	<0.001	0.113	45.70	0.120	0.264
FCR, g/g	32 (135)	2.23 (0.27)	−0.074 (−0.094; −0.054)	<0.001	<0.001	80.43	0.301	0.537

N: the number of studies; NC: the number of comparisons between the essential oil treatment and control treatment; SD: standard deviation; WMD: weighted mean differences between control and treatments with microalgae; CI: confidence interval of WMD; p-Value to χ² (Q) test of heterogeneity; I²: proportion of total variation of size effect estimates that is due to heterogeneity; ¹: Egger’s regression asymmetry test; ²: Begg’s adjusted rank correlation; ADFI: average daily feed intake; FCR: feed conversion ratio.

). However, the EP, EW, and EM increased in response to EO supplementation (p < 0.001). In contrast, the dietary inclusion of EOs decreased FCR (p < 0.001).

3.3. Egg Quality

Dietary supplementation with EOs increased the ET, ES, AH, HA, YC, and YI (p < 0.05;

Table 3. Egg quality of laying hens supplemented with essential oils.

Item	N (NC)				Heterogeneity		Egger Test 1	Begg Test 2
		Control Means (SD)	WMD (95% CI)	p-Value	p-Value	I2 (%)	p-Value	p-Value
Eggshell thickness (ET), mm	30 (106)	365.44 (68.85)	14.262 (10.811; 17.712)	<0.001	0.110	44.17	0.135	0.083
Eggshell strength (ES), kg/cm2	16 (66)	3.71 (1.04)	0.080 (0.052; 0.109)	<0.001	0.177	13.80	0.363	0.458
Albumen height (AH), mm	14 (44)	6.96 (0.76)	0.201 (0.115; 0.287)	<0.001	0.121	42.56	0.490	0.699
Haugh unit (HU)	29 (105)	76.85 (18.55)	1.102 (0.774; 1.431)	<0.001	0.209	42.18	0.109	0.230
Yolk color (YC)	21 (62)	7.37 (1.66)	0.071 (0.017; 0.124)	0.010	0.144	48.80	0.776	0.697
Yolk index (YI)	9 (32)	0.42 (0.04)	0.007 (0.001; 0.014)	0.030	<0.001	69.69	0.401	0.064
MDA in yolk, ng/g	5 (16)	2.11 (1.05)	−0.573 (−0.831; −0.315)	<0.001	<0.001	98.34	0.179	0.302

N: the number of studies; NC: the number of comparisons between essential oil treatment and the control treatment; SD: standard deviation; WMD: weighted mean differences between control and treatments with microalgae; CI: confidence interval of WMD; p-Value to χ² (Q) test of heterogeneity; I²: proportion of total variation of size effect estimates that is due to heterogeneity; ¹: Egger’s regression asymmetry test; ²: Begg’s adjusted rank correlation; MDA: malondialdehyde.

). However, the dietary inclusion of EOs decreased the MDA content in the yolk (p < 0.001).

3.4. Antioxidant Status

Table 4. Antioxidant status in the blood serum of laying hens supplemented with essential oils.

Item	N (NC)				Heterogeneity		Egger Test 1	Begg Test 2
		Control Means (SD)	WMD (95% CI)	p-Value	p-Value	I2 (%)	p-Value	p-Value
SOD, U/mL	5 (17)	64.74 (17.93)	1.147 (0.041; 2.253)	0.042	<0.001	84.66	0.064	0.121
GPx, U/mL	6 (20)	4283.00 (3183.00)	879.553 (506.015; 1253.091)	<0.001	<0.001	88.49	0.136	0.714
TAC, U/mL	4 (13)	5.17 (2.00)	1.163 (0.277; 2.049)	0.010	<0.001	87.71	0.639	0.418
MDA, nmol/mL	7 (20)	5.76 (2.689)	−0.324 (−0.523; −0.124)	0.001	0.231	47.66	0.554	0.189

N: the number of studies; NC: the number of comparisons between the essential oil treatment and control treatment; SD: standard deviation; WMD: weighted mean differences between control and treatments with microalgae; CI: confidence interval of WMD; p-Value to χ² (Q) test of heterogeneity; I²: proportion of total variation of size effect estimates that is due to heterogeneity; ¹: Egger’s regression asymmetry test; ²: Begg’s adjusted rank correlation; SOD: superoxide dismutase; GPx: glutathione peroxidase; TAC: total antioxidant capacity; MDA: malondialdehyde.

shows that EO supplementation increased the serum levels of SOD, GPx, and TAC (p < 0.05). In contrast, the dietary inclusion of EOs decreased the MDA content in blood serum (p = 0.001).

3.5. Intestinal Morphology

Table 5. Intestinal morphology of the laying hens supplemented with essential oils.

Item	N (NC)				Heterogeneity		Egger Test 1	Begg Test 2
		Control Mean (SD)	WMD (95% CI)	p-Value	p-Value	I2 (%)	p-Value	p-Value
Duodenum
Villus height (VH), μm	7 (20)	856.20 (295.90)	135.40 (62.57; 208.20)	<0.001	<0.001	99.11	0.329	0.413
Crypt depth (CD), μm	7 (20)	149.5 (83.50)	−12.63 (−19.22 −6.04)	<0.001	<0.001	97.69	0.428	0.330
Villus width (VW), μm	5 (15)	126.00 (56.90)	6.91 (−0.84; 14.66)	0.048	<0.001	84.39	0.348	0.324
VH/CD ratio	5 (14)	6.66 (2.97)	0.81 (0.56; 1.05)	<0.001	0.116	33.24	0.350	0.361
Jejunum
Villus height (VH), μm	6 (17)	656.30 (218.60)	112.95 (20.13; 205.77)	0.017	<0.001	99.42	0.367	0.065
Crypt depth (CD), μm	6 (17)	11.40 (59.90)	−3.97 (−7.46; −0.48)	0.026	<0.001	74.53	0.101	0.283
Villus width (VW), μm	4 (12)	88.10 (37.20)	11.48 (6.51; 16.45)	<0.001	<0.001	81.33	0.749	0.802
VH/CD ratio	5 (14)	7.47 (3.84)	1.72 (0.66; 2.78)	0.002	0.129	42.88	0.877	0.998
Ileum
Villus height (VH), μm	5 (15)	476.60 (90.60)	49.05 (21.27; 76.83)	<0.001	<0.001	93.91	0.101	0.170
Crypt depth (CD), μm	4 (12)	136.60 (23.16)	−16.91 (−24.55; −9.26)	<0.001	<0.001	68.83	0.727	0.073
Villus width (VW), μm	4 (12)	71.79 (18.01)	13.68 (8.83; 18.52)	<0.001	<0.001	76.95	0.100	0.431
VH/CD ratio	4 (12)	3.90 (1.07)	0.96 (0.62; 1.31)	<0.001	0.175	28.35	0.273	0.284

N: the number of studies; NC: the number of comparisons between the essential oil treatment and control treatment; SD: standard deviation; WMD: weighted mean differences between control and treatments with microalgae; CI: confidence interval of WMD; p-Value to χ² (Q) test of heterogeneity; I²: proportion of total variation of size effect estimates that is due to heterogeneity; ¹: Egger’s regression asymmetry test; ²: Begg’s adjusted rank correlation.

shows that dietary supplementation with EOs increased (p < 0.05) the VH, CD, VW, and VH/CD ratio in the duodenum, jejunum, and ileum.

3.6. Meta-Regression and Publication Bias

Table 2, Table 3, Table 4 and Table 5 show that the Egger regression asymmetry test was not significant (p > 0.05) for any of the response variables tested, indicating no publication bias.

On the other hand, Table 2 shows that there was heterogeneity (Q) (p ≤ 0.10) in the ADFI, EP, EW, and FCR. Likewise, a significant Q (p ≤ 0.10) was observed in YI and MDA in the egg (Table 3). In the blood serum, Table 4 shows a significant Q (p ≤ 0.10) in the concentration of SOD, GPx, and TAC. Finally, significant Q (p ≤ 0.10) was observed in the VH, CD, and VW in the duodenum, jejunum, and ileum (Table 5). However, Littell et al. [65] indicate that meta-regression analysis should only be applied when the variable of interest is reported in ten or more studies. Therefore, the meta-regression was only applied to the following response variables: ADFI, EP, EW, and FCR.

Table 6. Meta-regression of the effects of dietary essential oil supplementation on egg production and quality in laying hens.

Parameter		Hen’s Age	Breed/Strain	Supplementation Period	EOs Dose	Primary Bioactive Compound
Average daily feed intake (ADFI)	QM	0.006	41.842	0.614	1.422	9.509
	Df	1	7	1	1	9
	p-Value	0.939	<0.001	0.433	0.233	0.392
	R2 (%)	7.84	50.26	0.85	3.73	7.05
Egg production (EP)	QM	0.146	24.678	4.777	0.076	88.696
	Df	1	8	1	1	10
	p-Value	0.702	0.002	0.811	0.783	<0.001
	R2 (%)	0.00	12.95	7.05	0.00	37.99
Egg weight (EW)	QM	8.441	45.57	2.608	0.316	25.645
	Df	1	8	1	1	8
	p-Value	0.074	<0.001	0.106	0.574	0.001
	R2 (%)	3.41	34.42	0.00	0.00	32.02
Feed conversion ratio (FCR)	QM	0.005	56.77	3.452	0.891	99.485
	Df	1	8	1	1	10
	p-Value	0.943	<0.001	0.244	0.345	<0.001
	R2 (%)	0.00	14.81	0.00	0.00	29.99

QM: coefficient of moderators; QM is considered significant at p ≤ 0.05; df: degree of freedom; R²: the amount of heterogeneity accounted for; EOs: essential oils.

shows no significant relationship between the covariates hen’s age, supplementation period, and EO dose with any of the response variables tested. The breed/strain of the hens explained between 12.95 and 50.26% of the observed heterogeneity in the ADFI, EP, EW, and FCR. The primary bioactive metabolite covariate explained 37.99, 32.02, and 29.99% of the observed heterogeneity in the EP, EW, and FCR, respectively.

3.7. Subgroup Analysis

Figure 2a shows that the ADFI decreased (p < 0.01) when the laying hen breeds/strains used were Hy-Line White (WMD = −2.163 g/d) and Super Nick H&N (WMD = −2.590 g/d). In contrast, the ADFI increased when the breeds/strains of laying hens used were Hy-Line Brown (WMD = 0.631 g/d) and Bovans Brown (WMD = 3.081 g/d) and was not affected when other breeds/strains of laying hens were used (p > 0.05). On the other hand, the EP increased (p < 0.05; Figure 2b) when the breeds/strains of laying hens used were Hy-Line White (WMD = 2.526%), Hy-Line Brown (WMD = 1.766%), Lohmann Brown (WMD = 4.028%), ISA Brown (WMD = 5.196%), and Bovans Brown (WMD = 3.542%). However, the EP was not affected when other breeds/strains of laying hens were used (p > 0.05). Similarly, Figure 2c shows that the EW increased when the laying hen breeds/strains used were Hy-Line Brown (WMD = 0.287 g), Lohmann Brown (WMD = 0.688 g), Lohmann White (WMD = 0.561 g), ISA Brown (WMD = 1.561 g), and Bovans Brown (WMD = 3.449%). However, the EW was not affected when other breeds/strains of laying hens were used (p > 0.05). The FCR decreased (p < 0.01) when the breeds/strains of laying hens used were Hy-Line White (WMD = −0.085 g/g), Hy-Line Brown (WMD = −0.045 g/g), Super Nick H&N (WMD = −0.069 g/g), Lohmann Brown (WMD = −0.068 g/g), ISA Brown (WMD = −0.269 g/g), and Bovans Brown (WMD = −0.122 g/g). However, the FCR was not affected when other breeds/strains of laying hens were used (p > 0.05).

Figure 3a shows that the EP was increased (p < 0.01) when the primary bioactive metabolites of EOs were trans-anethole (WMD = 0.808%), terpinene-4-ol (WMD = 2.768%), cinnamaldehyde (WMD = 8.268%), blend (WMD = 2.650%), and menthol (WMD = 3.055%). However, when EOs were used with other primary bioactive metabolites, the EP was not affected (p > 0.05). On the other hand, Figure 3b shows that the EW increased (p < 0.05) when the primary bioactive metabolites of the EOs were trans-anethole (WMD = 0.122 g), cinnamaldehyde (WMD = 1.718 g), thymol (WMD = 0.282 g), blend (WMD = 0.677 g), and menthol (WMD = 1.671 g). However, the EW was not affected when EOs were used with other primary bioactive metabolites (p > 0.05). Figure 3c shows that the FCR decreased (p < 0.05) when the primary bioactive metabolites of the EOs were terpinene-4-ol (WMD = −0.100 g/g), blend (WMD = −0.097 g/g), and menthol (WMD = −0.074 g/g). However, when EOs were used with other primary bioactive metabolites, the FCR was not affected (p > 0.05).

4. Discussion

4.1. Performance

Some authors [11,12,13] have indicated that EOs can be used as additives to improve the flavor and palatability of feed for farm animals. However, in the present meta-analysis, EO supplementation did not affect the ADFI. In a previous meta-analysis, Irawan et al. [15] also did not observe changes in ADFI in broilers supplemented with EOs. On the other hand, higher EP, EW, EM, and lower FCR were observed in response to supplementation with EOs. In the present study, EO supplementation increased the SOD, GPx, and TAC serum levels. Furthermore, Cheng et al. [10] reported up to 18 and 20% higher serum concentrations of immunoglobulin G and interleukin-2 in laying hens supplemented with EOs. Likewise, previous studies [2,49] also detected that dietary supplementation with EOs decreased the count of bacteria Salmonella sp. and Escherichia coli in the small intestine and cecum of laying hens between 17 and 26%. These effects of EOs could improve the health status of laying hens and positively modify the EP, EW, EM, and FCR.

In laying hens, it has been reported that the dietary inclusion of EOs increases the secretion of digestive enzymes (chymotrypsin, lipase, and α-amylase) [2,24] and the digestibility of dry matter, crude protein, and ether extract [23,38]. In the present study, increased VH was observed in the duodenum, jejunum, and ileum in response to EO supplementation. Furthermore, He et al. [42] detected that EO supplementation increased the expression of glucose transporters (GLUT2) and peptides (PEPT1) in the duodenum and jejunum of laying hens. These effects of EOs could increase the absorption and metabolic availability of nutrients and benefit the EP, EW, EM, and FCR. On the other hand, previous studies [35,42] have indicated that in laying hens, the dietary inclusion of EOs (between 24 and 100 mg/kg DM) increases the cecal abundance of the genus microbial Lactobacillus, which has a negative correlation with FCR in laying hens [39]. Furthermore, recent studies [1,39] detected that, in laying hens, supplementation with EOs increased the relative cecal abundance of Anaerofilum, Fusobacterium, and Sutterella bacteria, which have positively correlated with EP in laying hens [39]. Consequently, similar effects of the consumption of EOs in the present study partially explain the positive effects observed in EP and FCR.

4.2. Egg Quality

Egg quality is important in laying hen production systems because it relates to consumer acceptability and preference [59] as well as economic profitability [18]. In particular, ET and ES are used as indicators to evaluate the quality of the eggshell [10], which influences the transport and storage of eggs [66]. In the present study, ET and ES increased in response to EO supplementation. These results are positive, since higher ET and ES could result in a lower rate of broken eggs [59]. In laying hens, recent studies [10,23] have reported that low doses (between 50 and 75 mg/kg feed) of EO mixtures increase intestinal calcium absorption by up to 14.7%. Similar effects of the consumption of EOs in the present study partially explain the observed increase in ET since, in laying hens, ET increases linearly with the increase in the metabolic availability of calcium. On the other hand, the higher ES observed in the present study could be related to the observed increase in ET since there was a positive correlation (r = 0.64) between ES and ET in eggshells from laying hens [67].

Malfatti et al. [66] indicated that AH, HU, YC, and YI are important parameters that serve as indicators of the internal quality of the egg. Particularly, AH, HU, and YI are indicators commonly used to assess egg freshness [66,68] because they have a negative correlation (r between 0.82 and 0.89) with their storage time [69]. Likewise, YC is an important parameter because most consumers prefer eggs with darker yolks [70]. In the present study, EO supplementation increased the AH, HU, YC, and YI, indicating that EOs can be used as a nutritional strategy to improve the color and internal quality in the eggs of laying hens. EOs contain terpenoids such as thymol and carvacrol [9,71], which have been reported to stimulate ovomucin synthesis in laying hens [72]. Similar effects of the consumption of EOs in the present meta-analysis would partly explain the increases observed in the AH, HU, and YI because, in laying hens, the AH, HU, and YI are positively correlated (r between 0.69 and 0.71) with the ovomucin concentration in egg [73]. On the other hand, Kavan et al. [43] mentioned that dietary pigments are the main factors influencing YC changes. Therefore, the higher YC observed in the present study suggests that the EOs used probably contained pigments.

There is limited information on the use of EOs to increase the oxidative stability in eggs from laying hens [33]. In the present study, a lower MDA content was observed in the egg yolk in response to dietary supplementation with EOs. This result indicates that EOs decrease yolk lipid peroxidation [54], which could increase the shelf life of eggs. Previous studies [74,75] have indicated that EOs contain various bioactive compounds with antioxidant properties that can be absorbed in the laying hens’ intestines and subsequently enter the egg yolk through blood circulation. This mechanism of absorption of the antioxidant compounds in the EOs partially explains the lower content of MDA in the yolk observed in the present study.

4.3. Antioxidant Status

In laying hens, ROS are continuously produced as a consequence of normal biological processes [22]. However, excessive accumulation of ROS can lead to OS [3,11]. According to Gholami-Ahangaran et al. [74], EOs can be used as natural antioxidants in poultry diets since they contain several bioactive metabolites with antioxidant activity. Serum levels of SOD, GPx, TAC, and MDA are essential to assess the antioxidant status in laying hens [10]. In the present study, EO supplementation increased the serum levels of SOD and GPx. According to Surai et al. [3], an increase in the serum levels of SOD and GPx decreases the oxidative damage caused by ROS in poultry cell membranes. To our knowledge, the mechanism of action of EOs or their bioactive metabolites on the serum levels of SOD and GPx has not been studied in laying hens. However, terpenoid consumption has been documented to increase the rodents’ expression of genes encoding SOD and GPx [76]. EOs contain a wide variety of terpenoids (e.g., linalool, carvonene, and 1,8-cineole) [71]. Therefore, similar effects of EO consumption in the present meta-analysis could explain the observed increase in SOD and GPx.

On the other hand, a higher TAC was observed in response to supplementation with EOs. This effect indicates that EOs improve the overall antioxidant status in laying hens. In poultry, most of the antioxidant metabolites of EOs are bioavailable, since after being ingested, they can be absorbed in the intestine and later transferred to the bloodstream [74,75]. Therefore, this effect could be related to the higher TAC detected in the present meta-analysis, since the TAC values in blood serum increase when the absorption and bioavailability of the antioxidants consumed are high [77].

In the present meta-analysis, the inclusion of EOs in the diets of laying hens decreased the serum MDA levels. This effect indicates that EOs can be used successfully to decrease lipid peroxidation in the blood of laying hens because, according to Nielsen et al. [78], the serum concentration of MDA decreases when lipid peroxidation is low. In addition, EOs contain several monoterpenes (e.g., p-cymene, limonene, and α-pinene) [71] that can be absorbed and transferred to the blood, where they can subsequently act by eliminating ROS [74]. This mechanism of action partially explains the lower serum concentration of MDA observed in the present study because lipid peroxidation decreases when the presence of ROS is low [79].

4.4. Intestinal Morphology

In laying hens, small intestine morphology characteristics such as the VH, VW, CD, and VH/CD ratio are used to assess the health and nutrient absorptive capacity [10]. The present study showed that the dietary addition of EOs increased the VH, VW, and VH/CD ratio and decreased the CD in the duodenum, jejunum, and ileum of laying hens. In a previous meta-analysis, Irawan et al. [15] also observed a higher VH and lower CD in broilers supplemented with EOs. A higher VH is related to a higher enterocyte count and production of digestive enzymes [49]. Likewise, an increase in VH and VW indicates a greater nutrient absorption surface in the intestine [23]. On the other hand, intestinal crypts contain cells that produce mucus and replace damaged and aged cells [49]. Some authors [23,52] have mentioned that the activity and depth of the crypt increase when cell detachment in the intestine is high due to inflammation induced by pathogens and their toxins. In addition, a high VH/CD ratio is associated with better structural integrity of the intestinal epithelium and a greater area of nutrient absorption [24]. Therefore, the changes in the VH, VW, CD, and VH/CD ratio observed in the present study suggest that EOs improve the integrity and absorptive capacity of the intestinal epithelium in laying hens.

In poultry, the consumption of EOs increases the mRNA expression of tight junction proteins (occludins and cadherins) [59] and decreases the count of pathogenic bacteria (E. coli and Salmonella sp.) in the small intestine [15,42]. According to Mousavi et al. [49] and Wang et al. [59], these effects reduce pathogen damage to intestinal epithelium cells and improve their integrity, which could partially explain the positive effects observed in the present study for the VH, VW, CD, and VH/CD ratio.

5. Conclusions

The results of this meta-analysis indicate that the inclusion of essential oils in the diets of laying hens can be used as a nutritional strategy to improve the productive performance, egg quality, antioxidant status in blood serum, and intestinal morphology. The best production and egg weight results were obtained with Lohmann Brown, ISA Brown, or Bovans Brown laying hens and when the primary bioactive compound of the essential oils was menthol, cinnamaldehyde, or mixtures of bioactive compounds. Likewise, the best feed conversion ratio was obtained with ISA Brown or Bovans Brown laying hens and when the primary bioactive compound of the essential oils was menthol, terpinen-4-ol, or mixtures of bioactive compounds.