The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis

Background: Due to their antimicrobial properties and safety, essential oils are currently proposed as a sustainable option for antibiotic alternatives in the livestock sector. This current systematic review and meta-analysis investigated the effects of dietary essential oil supplements on dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR) of small ruminants. Methods: A total of 12 studies (338 small ruminants) were included in this meta-analysis. The overall effect size was quantified using Hedges’ g with 95% confidence interval (CI) using a fixed-effect model. Publication bias was inspected using Begg’s and Egger’s tests, followed by trim and fill method to detect the number of potential missing studies. Results: Insignificant heterogeneity among studies was detected both on DMI ( P of Q = 0.810; I-square = 0.00%), ADG ( P of Q = 0.286; I-square = 17.61%), and FCR ( P of Q = 0.650; I-square = 0.00%). The overall effect size showed that essential oils supplementation had no significant impact on DMI (Hedges’ g = -0.12; 95% CI = -0.50 to 0.26; P = 0.429) and FCR (Hedges’ g = -0.17; 95% CI = -0.55 to 0.22; P = 0.284), but had a significant positive impact on ADG (Hedges’ g = 0.44; 95% CI = 0.12 to 0.76; P = 0.002). The result of publication bias analysis showed that DMI, ADG, and FCR did not present any significant biases ( P > 0.10), and no potential missing studies detected. Conclusions: Dietary essential oil could improve ADG of small ruminants, without any alteration on DMI and FCR. Further research in this topic is still required to provide stronger evidence of the potency of essential oil as a growth promoter for small ruminants.


Introduction
In animal nutrition, antibiotics become the first choice of feed additive due to their substantial benefit toward health and productivity. However, the routine use of this chemical additive yields residues in livestock products, and is also responsible for the development of microbial antibiotic resistance 1,2 . These factors represent a dangerous risk to human health, which has led to the global drive to reduce antibiotic use in the livestock sector. As a result, several natural products have been proposed to be used as antibiotic alternatives 3,4 .
Among natural feed additives, essential oils have a unique mechanism of action in livestock production. They can manipulate rumen fermentation characteristics 5,6 and subsequently improve growth rate 7,8 . However, other findings showed no meaningful effect of this feed additive on productive performance 9,10 , while another study showed a negative impact 11 . The inconsistent results among studies requires an appropriate tool to quantify the overall effect. Therefore, this study was conducted to measure the quantitative effects of dietary essential oil supplementation on dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR) of small ruminants using a systematic review and meta-analysis approach.

Methods
The systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline 12 . A completed PRISMA checklist is available in Reporting guidelines 13 .

Eligibility criteria
The inclusion and exclusion of the study were based on participants, interventions, comparisons, outcomes, and study design (PICOS) criteria as indicated in Table 1. Additionally, only publications written in English which was included in this study. All dates up until the date last searched were included.

Literature search strategy
The literature search was carried out using the following electronic databases: Scopus, PubMed, and SciELO. The search was last performed on 30 April 2020. Table 2 shows the full electronic search strategy.

Study selection
Results from the search were firstly checked for duplicates. After duplicate studies were removed, the titles and

Amendments from Version 1
The manuscript was revised according to the comments from reviewers with the following details: 1. Indicated original words before using abbreviations.
2. Deleted the statement regarding the authors' duties in study selection and data extraction.
3. Included the discussion about why essential oils can be used as a growth promoter for small ruminants 4. Added data about the type of ration and experimental design in Table 3.
Any further responses from the reviewers can be found at the end of the article REVISED abstracts were screened using the eligibility criteria (Table 1). Full texts of the selected studies were then further examined to find eligible studies. The authors of the included studies were not contacted for further clarification.

Data extraction
Data extracted included the following items: 1) authors; 2) animal species; 3) number of animals; 4) type of ration; 5) essential oil source; 6) experimental design, and 7) growth response variables. Growth response variables consisted of DMI, ADG, and FCR. Standard error or standard error of means were converted into standard deviation 14 . The data was pooled when a study used more than one dose of essential oils or tested both sexes of experimental animals 15 .

Effect size quantification
The overall effect size was quantified using Hedges' g 16 using a fixed-effect model. This model was chosen due to the insignificant heterogeneity among studies after checked using Cochran's Q 16 and I-square 17 .

Publication bias analysis
Publication bias was inspected using Begg's 18 and Egger's tests 19 , with P <0.10 set to determine the existence of publication bias. The trim and fill method 20 was employed to detect the number of potential missing studies and to adjust the overall effect size. All meta-analysis procedures were performed using Meta-Essentials version 1.4 21 . Figure 1 shows the PRISMA flow diagram. A total of 137 records were identified through database searching. Of these, 12 studies were eligible for the current meta-analysis. The essential oil sources included oregano 11,22-24 , thyme 25,26 , chavil 27 , juniper 7,28 , and mixed product 9,29 . Unfortunately, one study did not define the source of essential oil 8 . The main characteristics of the included studies are shown in Table 3. Extracted data of outcome measures is available as Extended data 30 .

Results
Data of ADG from two studies 11,25 were considered as outliers because their standardized residual was >|3| and thus were excluded from effect size quantification. Insignificant heterogeneity among studies was detected both for DMI (P of Q = 0.810; I-square = 0.00%), ADG (P of Q = 0.286; I-square = 17.61%), and FCR (P of Q = 0.650; I-square = 0.00%). As can be seen in Figure 2, the overall effect size showed that essential oil supplementation had no significant impact on DMI (P = 0.429) and FCR (P = 0.284), but had a significant positive impact on ADG (P = 0.002). The result of publication bias analysis showed that DMI, ADG, and FCR did not present any significant biases (P >0.10) ( Table 4). The trim and fill method also did not detect any potential missing studies for all parameters.

Discussion
The current meta-analysis showed that dietary essential oils significantly increased ADG of small ruminants. This finding probably related to the antimicrobial activity of essential oils, which could reduce ruminal protozoa population 31,32 . Protozoa population may represent up to 50% of the total biomass of rumen microbes 33 . They have a negative impact on nitrogen utilization by ruminants because they engulf and digest bacteria, thus reducing microbial protein flow to abomasum 34 . Additionally, the presence of protozoa is also associated with methane production, which is responsible for the loss of up to 12% of gross energy intake by ruminants 35 . Thereby, the reduction of the ruminal protozoa population by essential oil could increase microbial protein, as well as energy supply, which ultimately could improve the growth rate of small ruminants.
This study provides insight of the potency of essential oil as a growth promoter for small ruminants. However, the current findings should be interpreted with caution due to the limited data available. Moreover, the literature search only covers published literature, which could lead to publication bias. For that reason, further research in this topic is highly encouraged to provide stronger evidence.

Conclusions
The current meta-analysis reveals that dietary essential oil could improve ADG of small ruminants, without any alteration   on DMI and FCR. However, further research in this topic is still highly recommended to provide more robust evidence.

Data availability
Underlying data All data underlying the results are available as part of the article and no additional source data are required.
This project contains extracted data of outcome measures (dry matter intake, average daily gain, and feed conversion ratio). I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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