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Article

Dietary Oregano Oil Supplementation Improved Egg Quality by Altering Cecal Microbiota Function in Laying Hens

by
Lili Xian
1,2,
Yan Wang
1,2,
Da Peng
1,2,
Lei Zang
1,2,
Yidan Xu
3,
Yuanyuan Wu
4,
Jingjing Li
3,* and
Jing Feng
1,2,*
1
Institute of Animal Husbandry and Veterinary Medicine, Xizang Academy of Agricultural and Animal Husbandry Science, Lhasa 850009, China
2
Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lhasa 850009, China
3
School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China
4
Weifang Academy of Agricultural Sciences, Weifang 261071, China
*
Authors to whom correspondence should be addressed.
Animals 2024, 14(22), 3235; https://doi.org/10.3390/ani14223235
Submission received: 29 September 2024 / Revised: 9 November 2024 / Accepted: 9 November 2024 / Published: 12 November 2024
(This article belongs to the Section Poultry)
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Simple Summary

We investigated the effects of dietary oregano oil supplementation with different concentrations on the egg quality, intestinal morphology and cecal microbiome of late-phase laying hens. The results showed that dietary oregano oil supplementation could enhance eggshell thickness and increase the content of PUFA, thiamine, riboflavin, selenium and phosphorus in egg yolk. Meanwhile, oregano oil supplementation had a good effect on the intestinal morphology and structure of laying hens in the late laying period. The differential microorganisms were significantly correlated with egg quality characteristics. Oregano oil may enhance the amino acid content of eggs by increasing the abundance of Synergistota, which in turn enhances egg quality. These results will support the use of oregano oil as a new feed additive to improve egg quality. Overall, 25 mg/kg of dietary oregano oil had the best effects.

Abstract

Improving egg quality is one of the main objectives of the poultry industry. The objective of this study was to investigate the effects of dietary oregano oil supplementation with different concentrations on the egg quality, intestinal morphology and cecal microbiome of late-phase laying hens. A total of 300 55-week-old Snowy white laying hens were randomly divided into five groups and fed a basal diet (control) or basal diets supplemented with oregano oil at 25, 50, 75 and 100 mg/kg (O25, O50, O75 and O100) over a 100-day period. Compared with the control group, eggshell thickness and eggshell weight were increased significantly in eggs when oregano oil was added (p < 0.05). The highest content of polyunsaturated fatty acids (PUFAs) and C18:2n6c was observed in yolks with 25 mg/kg supplement among 5 groups (p < 0.05). The highest average content of riboflavin in egg yolk and thiamine in egg white was observed in the O25 group (p < 0.05). When the supplemental levels of oregano oil were 25 mg/kg and 50 mg/kg, the selenium and phosphorus content in the egg was significantly increased (p < 0.05). The ratio of villus height to crypt depth (V/C) and villus height of the small intestine was significantly increased compared with the chickens fed the basal diet (p < 0.05). Compared with the control group, the abundance of Megamonas was increased in the O50 group (p < 0.05). The unclassified_o__Bacteroidales in the O25 group were significantly higher than those in the other four groups (p < 0.05). These differential microorganisms were significantly correlated with egg quality characteristics. Dietary supplementation of oregano oil can significantly improve egg quality via gut microbiota alteration in laying hens.

1. Introduction

Eggs provide a cheap and easily accessible source of dietary protein and occupy a prominent position in the global food market [1]. Consumers are becoming increasingly concerned about the quality and nutritional value of eggs. The proteins, vitamins, and minerals found in eggs provide comprehensive benefits to maintain human health [2]. Egg quality is determined based on genetics and environmental conditions. Madalena et al. found that the eggs produced by commercial hens are more rounded, and the shells and yolks are darker than those of the four local breeds in Portugal [3]. Vlčková et al. investigated that storage time, the age of the hens and their interactions significantly affected egg weight, Haugh unit score and albumen pH [4]. Zhang et al. revealed that adding different concentration of vegetable oil to the diet could effectively improve egg quality [5].
Currently, phytogenic additives are recommended as safe and environmentally friendly alternatives and are widely used in the feed industry. Oregano oil is extracted from oregano, a perennial medicinal herb. It has a wealth of pharmacological properties, including antioxidant, antibacterial and anticancer properties [6,7]. It is one of the feed additives approved by the Ministry of Agriculture of China, with the characteristics of safety and high efficiency [8]. Therefore, it has been widely used in humans, mice, fish and chicken [9,10,11,12]. In poultry production, the beneficial effects of oregano oil have also been explored. Oregano at 1.0% enhanced productive performance, fecundity and hatching ability, and positively influenced the oxidative stability of shell eggs during storage [13]. Swanny et al. showed that the addition of 150 ppm oregano oil to the diet improved the percentage of egg production, yolk color, shell thickness and shell color [14]. Fei et al. suggested that egg production performance, feed conversion ratio, fatty acid content and microbial composition of eggs from late-laying hens could be improved by supplementation with 200 mg/kg oregano oil and 20 mg/kg cinnamaldehyde [15].
Egg quality in late-laying hens deteriorates due to impaired gut function, immune imbalance and gut microbiota imbalance caused by high-intensity production [16,17]. At the same time, the gut is the largest compartment of the immune system, and is involved in the protection of the host by providing a strong defense against invasion from the external environment [18]. The cecum of chickens harbors a complex microbiome [19,20]. Although several studies showed that dietary oregano oil supplementation could exert favorable effects on gut health in broilers [15,16,17], it is unknown whether oregano oil could improve egg quality by altering intestinal microbiota composition in later-laying hens. Therefore, the aims of this study were to investigate the effects of dietary oregano oil supplementation with different concentrations on the egg quality, intestinal morphology and cecal microbiome of late-phase laying hens.

2. Materials and Methods

2.1. Animals and Design

The trial was conducted at the Tibetan chicken-breeding base of the Tibet Autonomous Region Academy of Agricultural and Animal Husbandry Sciences. A total of 300 55-week-old snowy white chickens with similar body weight and good health were randomly divided into five groups that were further allocated to six replicates. Each replicate consisted of 10 broilers that were housed in cages. The dietary groups were as follows: the control group (group C) was fed a basal diet, and the experimental groups (groups O25, O50, O75 and O100) were fed a basal diet supplemented with oregano oil of 25, 50, 75 and 100 mg/kg, respectively. The oregano oil was purchased from Beijing Fedi Feed Technology Co., Ltd., Beijing, China. According to the “Feeding Standard for Chickens” (NY/T 33-2004) [21], the composition and nutritional level of the experimental basal diet are shown in Table 1. The experiment used 16 h of light and 8 h of darkness, and the light began at 06:00. Feed and water were provided ad libitum during the experimental period. The same environmental and management standards were applied to all birds. For specific feeding specifications refer to “Technical Specifications for Breeding of Lhasa White Chicken (Breed Group)” (DB54/T 0036-2009) [22].

2.2. Sample Collection

After a 100-day trial period, five eggs per replicate were randomly collected for quality parameter measurement. In addition, six chickens per group were randomly selected and transported to the slaughterhouse for sample collection. The duodenum, jejunum and ileum were sampled for intestinal morphology (n = 6). Intestinal segments were gently flushed with ice-cold phosphate-buffered saline (PBS, pH  =  7.4) to remove intestinal contents and placed in 4% paraformaldehyde for fixation. The contents of the cecum were then rapidly frozen in liquid nitrogen and stored at −80 °C until 16S rRNA sequencing (n = 6).

2.3. Egg Quality Parameters

Excluding abnormal eggs and double-yolked eggs, the following egg quality parameters were evaluated. Egg weight, yolk weight, and eggshell weight were measured using an electronic balance. Yolk thickness, eggshell thickness and air chamber diameter were measured by vernier caliper. The length and short diameter of the eggs were also measured by vernier caliper. The egg shape index was calculated using the formula egg shape index = length diameter of egg/short diameter of egg. The Haugh units (HU) were obtained using an egg quality analyzer (EMT-7300II, Robotmation, Tokyo, Japan).
The hydrolytic amino acid profile in the egg was measured according to the China National Standard (GB 5009.124-2016) [24]. The different tastes of amino acids could be divided into delicious amino acids (including aspartic acid and glutamic acid), sweet amino acids (including serine, glycine, proline, and alanine), and bitter amino acids (including lysine, valine, isoleucine, leucine, phenylalanine, histidine, arginine, and tyrosine). The free fatty acids in the egg were measured based on the method described by the China National Standard (GB/T 5009.168-2016) [25]. Riboflavin and thiamine were measured using high performance liquid chromatography following the method in the China National Standard (GB 5009.85-2016) [26] and (GB 5009.84-2016) [27], respectively. Glucose, total soluble sugar, total cholesterol, and triglyceride contents were measured using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Crude protein was measured using kjeldahl determination, as described by the China National Standard (GB 5009.5-2016) [28], and crude fat was measured using soxhlet extraction, as described by the China National Standard (GB 5009.5-2016). The moisture proportion was calculated by drying and weighing. The ash proportion was calculated by burning and weighing. The potassium, calcium, phosphorus, magnesium, sodium, iron, selenium and zinc concentrations were determined by inductively coupled plasma-mass spectrometry according to the China National Standard (GB 5009.268-2016) [29,30].

2.4. Morphology of Small Intestine

After fixation for 24 h, the intestinal samples were dehydrated using ethanol and cleared with xylene, and then the samples were embedded in paraffin. Cross sections were made at a thickness of 5 μm and stained with hematoxylin-eosin. Morphological examination was applied at 40× magnification through an optical microscope (Nikon Eclipse Ci-L, Tokyo, Japan). Villus height and crypt depth of five intact well-oriented villi per segment were measured for each intestinal cross section using Image-Pro Plus 6.0 (Media Cybemetics, Rockville, MD, USA). The villus height to crypt depth ratio was then calculated. The average of the values for each cross section was used for further analysis.

2.5. Sequencing and Analysis of 16S rRNA Gene

Microbial DNA was extracted from the cecal samples using the E.Z.N.A.® DNA kits (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocol. On a 1% agarose gel, the concentration and purity of DNA were detected. The barcoded fusion forward primer 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3 and V4 hyper variable region of the 16S rRNA gene. High-throughput sequencing was conducted on an Illumina MiSeq PE300 platform by Beyotime Biotechnology Company (Shanghai, China).
The reads of each sample were demultiplexed, quality controlled by FATSP (version 0.12.0) and merged using Flash software (version 1.2.7). The UPARSE algorithm was used to cluster sequences into operational taxonomic units (OTUs) at a 97% similarity level. The OTUs were annotated based on Silva and UNITE taxonomy databases. QIIME software (https://qiime2.org) was utilized to generate species abundance at the phylum and genus levels. The R (version 4.1.3) was used to perform general statistical analysis and visualize results. Alpha diversity was estimated using the ACE, Chao1, Shannon and Simpson indices. Principal Coordinates Analysis (PCoA) was conducted to assess the differences in beta diversity between groups. Linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed with a p-value < 0.05 for the Kruskal–Wallis test to compare the relative abundance of different taxa between groups.

2.6. Statistical Analysis

Statistical analysis was performed with SPSS 299.0 software (Chicago, IL, USA). One-way analysis of variance (ANOVA) was performed with Duncan’s post hoc test when the homogeneity of variance is significant (p < 0.05). All the analysis results were considered as the indication of statistical significance at p < 0.05.

3. Results

3.1. Egg Quality Parameters

The effects of different levels of oregano oil on egg quality are presented in Table 2. A significant difference was observed for eggshell thickness, air chamber diameter and eggshell weight (p < 0.05). The largest average air chamber diameter is the egg with 50 mg/kg oregano oil supplement (p < 0.05). Eggshell thickness and eggshell weight were increased significantly in eggs when oregano oil was added (p < 0.05). However, egg weight, yolk weight, Haugh unit, egg white thickness and yolk thickness did not differ significantly between the control and treatment groups (p < 0.05).

3.2. Amino Acids Profile

Data on the effect of oregano oil supplementation on the amino acid profile of eggs are shown in Table 3. A lack of statistical significance was observed for delicious amino acids, sweet amino acids and bitter amino acids in egg white and yolk (p > 0.05). The total essential amino acid (EAA) content in egg yolk was significantly lower in the O25 group than that of the other four groups (p < 0.05). However, there was no significant effect of the diet treatment on total EAA and individual EAA in egg white.

3.3. Fatty Acid Content

The levels of fatty acids in egg yolk are presented in Table 4. A total of 15 fatty acids were detected in yolk. Although the O50 group showed the greatest reduction in C4:0 content (p < 0.05), there was no significant difference in total saturated fatty acids and monounsaturated fatty acids in yolk (p > 0.05). The highest content of polyunsaturated fatty acids (PUFAs) and C18:2n6c were observed in yolk in the O25 group (p < 0.05).

3.4. Chemical Properties

The results of the present study highlighted the effect of different levels of oregano oil on chemical properties eggs (Table 5). There were significant differences in thiamine content in egg white and riboflavin content in yolk among all groups (p < 0.05). The highest average content of riboflavin in egg yolk and thiamine in egg white were observed in the O25 group (p < 0.05). There were significant differences in total soluble sugar in eggs among all groups (p < 0.05). When the concentration was 50 mg/kg, the total soluble sugar content in egg white and yolk was the highest (p < 0.05).

3.5. Mineral Elements

The mineral elements of eggs from various oregano oil supplementations are shown in Table 6. In egg white, the results revealed that groups treated with oregano oil had significantly increased phosphorus content (p < 0.05). When the supplemental levels of oregano oil were 25 mg/kg and 50 mg/kg, the selenium content in egg yolk was significantly increased (p < 0.05), but when the supplemental level of oregano oil was 75 mg/kg or above, the selenium level in egg yolk would start to drop. Oregano oil supplementation had no effect on potassium, calcium, magnesium, sodium, iron and zinc content (p > 0.05).

3.6. Intestinal Morphology

The morphological characteristics of intestinal tissues obtained from chickens in the different treatment groups are shown in Figure 1. All animals fed 0 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg and 100 mg/kg of oregano oil presented normal ranges for the heights and structures of villi and intestinal crypts. The villus height of the duodenum and ileum supplemented with oregano oil was significantly increased compared with the chickens fed the basal diet (p < 0.05) (Figure 1B). When oregano oil was added at concentrations of 50 mg/kg and 75 mg/kg, the crypt depth of the jejunum significantly decreased (p < 0.05). There was no significant effect of oregano oil on the crypt depth of the duodenum and ileum (p > 0.05) (Figure 1C). The ratio of villus height to crypt depth (V/C) of the duodenum, jejunum and ileum was significantly increased compared with the chickens fed the basal diet (p < 0.05) (Figure 1D). The results indicated that different levels of oregano oil supplementation in the basal diet had a good effect on the intestinal morphology and structure of snowy white chickens.

3.7. Cecum Microbial Diversity

A total of 644 OTUs were identified in all five cecum groups, whereas seven, zero, one, zero, and four specific operational taxonomic units (OTUs) were observed in the C, O25, O50, O75 and O100 groups, respectively (Figure 2A). The addition of oregano oil to the diet had no significant effect (p > 0.05) on the Chao1, Shannon, and Simpson indexes of bacterial richness and diversity (Figure 2B–D). ANOSIM analysis is a non-parametric test, based on the Bray–Curtis algorithm, which helps to determine whether differences between groups are observably greater than within group variations (Figure 2E). In this case, the R value can vary between −1 and 1, with values = 0.058 indicating significant between-group differences. The PCoA plot showed that there was no obvious separation among the five groups (Figure 2F).
At the phylum level, the cecal microbiota of the five groups was mainly composed of Bacteroidetes, Firmicutes, WPS-2, Desulfobacterota and Proteobacteria (Figure 3A). Among them, Firmicutes and Bacteroidetes occupied the largest proportion. There were no significant differences among all groups at the phylum level (p > 0.05). Analysis of the gut microbiota at the genus level showed that groups supplemented with 50 mg/kg oregano oil had an increased abundance of Megamonas compared with that in the control group (p < 0.05) (Figure 3B). The unclassified_o_Bacteroidales in the O25 group were significantly higher than those in the other four groups (p < 0.05). The abundance of Phascolarctobacterium was higher in the O75 group than that in the other four groups (p < 0.05). There was no difference in the abundance of Bacteroides and Rikenellaceae_RC9_gut_group (p > 0.05).
The linear discriminant analysis (LDA) effect size results showed that 21 genera were markers that distinguished the three groups of samples (Figure 3C). Five genera were greatly enriched in the C group: s_norank_g_UCG-008, g_UCG-008, s_norank_f_Rikenellaceae, g_norank_f_Rikenellaceae, and s_Alistipes_inops. Only two genera were enriched in the O25 group: s_norank_g_Paludicola and g_Paludicola. Three genera were significantly associated in the O50 group: s_norank_g_Sellimonas, s_norank_g_Solobacterium and g_Solobacterium. Three genera were enriched in the O75 group: s_norank_f_Tannerellaceae, g_norank_f_Tannerellaceae and s_Lactobacillus_ingluviei. Eight genera were significantly associated in the O100 group: s_norank_p_Proteobacteria, g_norank_p_Proteobacteria, f_norank_p_Proteobacteria, o_norank_p_Proteobacteria, c_norank_p_Proteobacteria, s_norank_g_Butyricimonas, g_Butyricimonas and f_Enterobacteriaceae.

3.8. Correlational Analysis Between Cecal Microbiota and Egg Quality

The correlations between cecal microbiota and egg quality parameters were analyzed (Figure 4). In egg yolk, correlation analysis revealed that the riboflavin, PUFA and C18:2n6c were negatively correlated with the abundance of Synergistota, whereas leucine, methionine and total EAA content were positively correlated with the abundance of Synergistota (p < 0.05). The findings suggest that oregano oil may enhance the amino acid content of eggs by increasing the abundance of Synergistota, which in turn enhances egg quality. Dietary Synergistota supplementation may be an effective strategy to improve egg quality in the poultry industry. Riboflavin was negatively correlated with Fusobacteriota (p < 0.05). Total EAA showed a positive correlation with Desulfobacterota (p < 0.05). In egg white, there was a negative correlation between thiamine and the abundance of Desulfobacterota and Synergistota (p < 0.05). The abundance of Fusobacteriota, Synergistota and Proteobacteria was positively correlated with total phosphorus (p < 0.05).

4. Discussion

Since oregano oil has been shown to have antioxidant, antibacterial and anticancer properties, it is widely used in the poultry industry [6,7,31]. In this study, the addition of oregano oil to the diet improved eggshell thickness in the late-laying period, which is consistent with previous reports [32,33]. Eggshell thickness, as an important index of egg quality, is important for egg transportation and storage [34]. Egg shell powder is considered a good source of highly bioactive calcium. This beneficial effect could be attributed to the active ingredients (i.e., thymol and carvacrol) in oregano oil, which improved intestinal health and nutrient utilization [33,35]. Amino acids play an important role in human nutrition and health. In this study, the effects of oregano oil on the amino acid composition of eggs were explored. Methionine and leucine are essential amino acids for protein synthesis in an animal body [36,37]. The results showed that a 25 mg/kg supplement of oregano oil reduced the content of methionine and leucine in the yolk. There was no significant effect of the other concentrations on essential amino acids.
Due to the close relationship between dietary lipids and the occurrence of diseases, the lipid composition of eggs has received much attention [38]. Previous studies presented the health benefits of polyunsaturated fatty acids (PUFA) on cardiovascular disease, diabetes, cancer, Alzheimer’s disease, dementia, depression and visual and neurological development [38,39,40]. In the present study, the PUFA content of group O25 and group O50 were significantly higher than that of the other three groups, implying the beneficial value of these two groups. The supplements of oregano oil changed the fatty acid content in egg yolk, probably by affecting serum cholesterol and lipid metabolism in the liver of laying hens [41,42,43,44,45]. Thiamine and riboflavin are water-soluble B-complex vitamins found in a variety of animal and vegetable products. They are found abundantly in lean pork, legumes, and eggs [46]. Adequate intake of riboflavin and thiamine can help preterm infants achieve adequate plasma concentrations and normal functional indices [47,48]. Riboflavin requirements appear to increase with exercise, dieting and dieting plus exercise in both young and older women engaged in moderate activity [49]. In this study, the highest average content of riboflavin was observed in the O25 group (p < 0.05). Therefore, consumption of eggs supplemented with 25 mg/kg oregano oil may have a positive effect on premature infants, pregnant women, kidney stone patients or athletes.
Phosphorus is a mineral that naturally occurs in many foods and is also available as a supplement. Phosphorus regulates the normal function of nerves and muscles [50]. The egg yolk contains 1% minerals, with phosphorus as the most abundant mineral component [51]. In our study, groups treated with oregano oil had significantly increased phosphorus content (p < 0.05). The essential trace mineral, selenium, is of fundamental importance to human health [52]. Se deficiency in humans increased the risk of some types of cancer and led to impaired mineralization of bones and teeth [53]. Humans generally increase their intake of Se by eating more Se-rich foods. Additionally, chicken eggs contain a large amount of selenium, and are an important source of selenium in the human diet [54]. In this study, the selenium content in yolk in group O25 and O50 was significantly increased (p < 0.05). In other words, consumption of eggs supplemented with 25 mg/kg and 50 mg/kg oregano oil may be helpful for people who are deficient in selenium and phosphorus.
Intestinal mucosal histology can be used to evaluate intestinal health [55]. Histological examination of the small intestine showed that the villus height and villus height/villus depth ratio were significantly increased when oregano oil was added. A previous study also suggested that oregano oil could increase the villus height and decrease the crypt depth, promoting the absorption of nutrients in the intestine [47]. At the same time, dietary supplementation of oregano oil and cinnamaldehyde could improve the performance of later-period laying hens, increase the villi absorption area, facilitate calcium absorption and increase eggshell thickness [33,48]. Thus, the oregano oil supplements probably increased thickness of the eggshell by improving intestinal morphology.
There are a large number of complex microbial flora in the intestinal tract of poultry, which can stimulate the growth and development of the gut, activate the immune system and promote the digestion and absorption of nutrients [56]. In the present study, there was no significant difference in the α-diversity among five groups. However, Feng et al. suggested that the microbiome composition was changed after oregano oil supplements [33]. Gao et al. also revealed that the combination of oregano oil and cinnamaldehyde increased the abundance of cecal flora and cecal microbial community structure [15]. The reason for this difference may be due to the fact that the concentration of oregano oil in this study is insufficient, and the combined effect of oregano oil and cinnamaldehyde is more obvious. The microbiota composition and specific taxa variation after the addition of oregano oil were then further analyzed. At the phylum level, the cecal microbiota was mainly composed of Bacteroidetes, Firmicutes, WPS-2, Desulfobacterota and Proteobacteria. Bacteroidetes are considered the leading factors for a healthy state, intestinal immunity, digestion and sophisticated homeostasis safeguarded by gut microbiota [57,58], while Desulfovibrio was considered to be harmful bacteria that inhibits the production of short-chain fatty acids [59,60]. In this study, Desulfobacterota was highly negatively correlated with thiamine and riboflavin in eggs. In addition, studies have shown that dietary C. butyricum supplementation could improve feed efficiency and yolk color [61]. Megamonas, hypermegale and Butyrivibrio species strains will improve immunity, thereby preventing and treating Behcet’s disease [62]. In this experiment, the content of Megamonas bacteria in the O50 group was significantly higher than that in other groups (p < 0.05), suggesting that supplementation of 50 mg/kg oregano oil may reduce the risk of Behcet’s disease.

5. Conclusions

In conclusion, the results of the present study indicate that dietary oregano oil supplementation could enhance eggshell thickness and increase the content of health-promoting PUFA, thiamine, riboflavin, selenium and phosphorus in egg yolk. Meanwhile, different levels of oregano oil supplementation in the basal diet had a good effect on the intestinal morphology and structure of laying hens in the late laying period. The dietary oregano oil supplementation regulated leucine, methionine, total EAA, riboflavin, C18:2n6c and PUFA content in egg yolk and thiamine and total phosphorus in egg white via cecal microbiota alteration. These results will support the use of oregano oil as a new feed additive to improve egg quality. Overall, dietary 25 mg/kg oregano oil had the best effects.

Author Contributions

Conceptualization, L.X. and Y.W. (Yan Wang); methodology, L.Z. and D.P.; resources, L.X., D.P. and Y.W. (Yuanyuan Wu); software, J.L. and Y.X.; writing-original draft, L.X.; writing-review and editing, J.L. and J.F.; project administration, J.F.; funding acquisition, J.L. and J.F. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by National laying hen industrial technology system (CARS-40-S19), the National key research and development plan (2022YFD1600901), Shandong Province technology innovation guidance program (YDZX2023002), Natural Science Foundation of Southwest University of Science and Technology (Grant No. 22zx7151), the Sichuan Science and Technology Program (Grant No. 2023NSFSC1147) and the Open Fund of Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province (Grant No. CNDK-2023-01).

Institutional Review Board Statement

The experimental procedures were approved by the Animal Care and Use Committee of the Institute of Animal Husbandry and Veterinary Science, Tibet Autonomous Region (approval number: XMS20220420).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be available upon request from the corresponding author.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Animals 14 03235 g001
Figure 1. Small intestine morphology in broiler supplemented with oregano oil based on hematoxylin and eosin staining observed under 100× magnification: (A) intestinal morphological structure; (B) villus height V (μm); (C) crypt depth (μm); (D) ratio of villus height to crypt depth V/C. The data are presented as a mean and standard error of the mean. Bars with different superscript letter are statistically different (p < 0.05).
Figure 1. Small intestine morphology in broiler supplemented with oregano oil based on hematoxylin and eosin staining observed under 100× magnification: (A) intestinal morphological structure; (B) villus height V (μm); (C) crypt depth (μm); (D) ratio of villus height to crypt depth V/C. The data are presented as a mean and standard error of the mean. Bars with different superscript letter are statistically different (p < 0.05).
Animals 14 03235 g001
Animals 14 03235 g002
Figure 2. (A) Venn diagram illustrating the overlaps of microbial OTUs between the five groups. Alpha diversity of the cecum bacteria between the five groups: (B) Chao1 index; (C) Shannon index; and (D) Simpson index. (E) Differences between ANOSIM groups. (F) PCoA of taxonomical classifications of ruminal bacteria communities based on UniFrac distances.
Figure 2. (A) Venn diagram illustrating the overlaps of microbial OTUs between the five groups. Alpha diversity of the cecum bacteria between the five groups: (B) Chao1 index; (C) Shannon index; and (D) Simpson index. (E) Differences between ANOSIM groups. (F) PCoA of taxonomical classifications of ruminal bacteria communities based on UniFrac distances.
Animals 14 03235 g002
Animals 14 03235 g003
Figure 3. (A) Relative abundance of bacteria community proportions at the phylum. (B) Relative abundance of bacteria community proportions at the genus. (C) Histogram of the linear discriminant analysis (LDA) effect among the three groups, and the LDA score (log10) > 2 were shown.
Figure 3. (A) Relative abundance of bacteria community proportions at the phylum. (B) Relative abundance of bacteria community proportions at the genus. (C) Histogram of the linear discriminant analysis (LDA) effect among the three groups, and the LDA score (log10) > 2 were shown.
Animals 14 03235 g003
Animals 14 03235 g004
Figure 4. (A) Pearson’s correlation analysis between the cecal microbiota and egg quality parameters in yolk. (B) Pearson’s correlation analysis between the cecal microbiota and egg quality parameters in white. Red represents the positive correlation, and green represents the negative correlation. * p < 0.05. ** p < 0.01.
Figure 4. (A) Pearson’s correlation analysis between the cecal microbiota and egg quality parameters in yolk. (B) Pearson’s correlation analysis between the cecal microbiota and egg quality parameters in white. Red represents the positive correlation, and green represents the negative correlation. * p < 0.05. ** p < 0.01.
Animals 14 03235 g004
Table 1. Comparison and chemical composition of the basal diet.
Table 1. Comparison and chemical composition of the basal diet.
Ingredients (%) Nutrient Levels 2
Corn62.70ME (MJ/kg)11.09
Soybean meal (CP 43%)26.30CP (%)16.61
CaHPO41.00CF (%)3.31
DL-Methionine0.10Available phosphorous, % (%)0.35
Limestone8.50Lysine (%)0.85
Choline chloride (70%)0.10Methionine (%)0.35
NaCl0.30Ca (%)3.50
1 Premix1.00Na (%)0.01
Total100
1 Premix, supplied each kg consists of: VA 10,000 IU, VD3 3000 IU, VE 20 IU, VK 32 mg, thiamine 1 mg, riboflavin 8 mg, calcium pantothenate 40 mg, niacin 32.5 mg, pyridoxine 8 mg, biotin 2 mg, folic acid 1.5 mg, VB12 5 mg, choline 500 mg, Mn 70 mg, I 1 mg, Fe 80 mg, Cu 8 mg, Zn 80 mg and Se 0.3 mg. 2 Nutrient levels, Calculated according to NRC (1994) [23].
Table 2. Effects of oregano oil on egg quality parameters.
Table 2. Effects of oregano oil on egg quality parameters.
ItemConcentration (mg/kg)SEMp Value
0255075100
Egg weight (g)52.0251.2154.0352.9752.400.510.53
Yolk weight (g)17.2316.3918.0517.5316.770.220.14
Eggshell weight (g)5.39 a6.38 b6.42 b6.38 b6.35 b0.120.01
Haugh unit72.0084.8383.3382.6679.001.600.07
Egg white thickness (mm)5.827.247.277.196.510.220.17
Yolk thickness (mm)17.1818.1118.6918.8418.000.210.08
Eggshell thickness (mm)0.25 a0.33 b0.33 b0.29 b0.34 b0.0110.00
Air chamber diameter (mm)16.01 a15.92 a17.21 b17.09 ab16.03 a0.2150.04
1 Egg shape index1.331.311.301.331.290.210.75
a,b Data within the same row marked with different small letters above the data indicate statistically significant differences (p < 0.05), while the same or no small letters indicate non-significant differences (p > 0.05). 1 Egg shape index = Ratio of length diameter of egg to short diameter of egg.
Table 3. Effects of oregano oil on amino acid composition of egg.
Table 3. Effects of oregano oil on amino acid composition of egg.
Items (mg/g)Concentration (mg/kg)1 SEMp Value 2
0255075100
Egg white
Glutamic acid14.1612.8112.6214.0813.560.450.79
Aspartic acid11.9510.9310.6011.8111.650.350.75
Glycine5.685.425.155.425.600.230.97
Alanine10.229.329.0810.289.770.320.76
Serine12.3911.3311.0612.4511.930.460.87
Proline5.465.115.315.695.230.230.96
Leucine0.210.150.160.180.190.010.23
Isoleucine13.8512.7012.7514.1113.230.540.92
Valine10.349.496.2210.679.810.770.41
Phenylalanine6.636.096.466.996.290.260.89
Tyrosine3.553.373.503.783.410.150.94
Histidine3.703.593.013.443.750.160.66
Lysine7.907.196.837.977.580.320.83
Arginine9.026.668.389.268.730.600.73
Methionine3.392.923.283.803.170.170.61
Threonine6.906.206.207.146.630.270.80
Cysteine6.816.196.487.116.450.270.87
Total EAA49.2144.7441.9150.8646.891.980.69
Delicious amino acids26.1123.7423.2225.8925.210.800.77
Sweet amino acids33.7531.1730.6033.8432.531.200.91
Bitter amino acids55.2049.2347.3256.3952.992.330.76
Yolk
Glutamic acid14.0013.6213.1413.3913.910.140.23
Aspartic acid11.1810.8710.3510.5110.910.120.13
Glycine7.987.457.527.697.960.090.21
Alanine9.809.589.009.009.650.140.17
Serine17.0216.4216.3816.8217.470.170.20
Proline9.599.449.6710.079.620.110.50
Leucine0.21 abc0.19 a0.20 ab0.22 bc0.23 c0.000.04
Isoleucine16.9516.4116.3416.6316.870.110.29
Valine12.0411.8511.8311.9112.010.060.85
Phenylalanine7.237.137.237.397.190.070.84
Tyrosine5.265.255.275.495.450.050.45
Histidine3.603.463.303.403.730.080.49
Lysine9.098.738.478.439.040.120.23
Arginine9.879.779.819.749.870.070.98
Methionine6.27 b1.93 a3.83 ab6.41 b6.22 b0.620.04
Threonine9.419.389.279.509.780.070.25
Cysteine9.409.089.099.349.390.060.29
Total EAA61.21 b55.63 a57.72 ab60.49 b61.33 b0.780.04
Delicious amino acids 325.1824.4923.4823.8924.820.250.17
Sweet amino acids 444.3942.8942.5743.5744.700.340.19
Bitter amino acids 564.2562.7962.4463.2164.381.230.49
a–c Data within the same row marked with different small letters above the data indicate statistically significant differences (p < 0.05), while the same or no small letters indicate non-significant differences (p > 0.05). 1 SEM: standard error means. 2 Overall treatment p-value. 3 Delicious amino acids = Aspartic acid + Glutamic acid. 4 Sweet amino acids = Serine + Glycine + Proline + Alanine. 5 Bitter amino acids = Lysine + Valine + Isoleucine + Leucine + Phenylalanine + Histidine + Arginine + Tyrosine.
Table 4. Effects of oregano oil on fatty acid composition of egg.
Table 4. Effects of oregano oil on fatty acid composition of egg.
Items (mg/L)Concentration (mg/kg)SEMp Value
0255075100
Yolk
C4:08.51 bc7.48 abc3.40 a5.19 ab10.58 c0.870.04
C6:08.758.025.835.529.470.570.07
C11:03.782.082.141.865.250.480.08
C14:04.7119.5915.653.691.162.830.13
C14:10.003.553.020.000.000.730.32
C16:0314.591246.78903.54259.6583.01182.400.21
C16:142.78168.30132.4330.9312.1126.510.25
C17:00.0010.288.143.250.001.540.08
C18:086.24375.21263.4887.7425.8452.700.17
C18:1n9c239.241080.65914.37227.2065.73180.400.28
C18:2n6c198.25 a1148.39 b875.38 b215.46 a51.08 a136.310.01
C20:20.008.546.702.170.001.350.12
C20:3n31.066.864.490.490.001.000.10
C20:4n615.9063.9763.6118.367.3111.570.36
C23:05.718.036.136.194.730.610.59
1 SFA432.291677.461208.31373.08140.04239.420.20
MUFA 2282.011252.501049.82258.1377.84207.540.28
PUFA 3215.21 a1227.75 b950.18 b236.48 a58.38 a148.480.01
a–c Data within the same row marked with different small letters above the data indicate statistically significant differences (p < 0.05), while the same or no small letters indicate non-significant differences (p > 0.05). 1 SFA: saturated fatty acid (SFA = C4:0 + C6:0 + C11:0 + C14:0 + C16:0 + C17:0 + C18:0 + C23:0). 2 MUFA: monounsaturated fatty acid (MUFA = C14:1 + C16:1 + C18:1n9c). 3 PUFA: polyunsaturated fatty acid (PUFA = C18:2n6c + C20:2 + C20:3n3 + C20:4n6).
Table 5. Effects of oregano oil on chemical properties contents of egg.
Table 5. Effects of oregano oil on chemical properties contents of egg.
ItemsConcentration (mg/kg)SEMp Value
0255075100
Egg white
Glucose (mg/g)4.263.933.493.993.990.190.82
Crude protein (%)12.4011.7913.0213.0211.850.340.70
Total soluble sugar (mg/g)7.42 a7.71 a9.74 b7.67 a7.79 a0.260.00
Crude fat (%)0.510.560.560.580.540.010.09
Ash content (%)0.720.730.720.690.660.010.60
Moisture (%)84.6985.2684.3283.8485.310.350.70
Total cholesterol (mg/g)0.130.100.330.300.240.050.47
Triglyceride (mg/g)1.200.960.880.260.320.140.12
Riboflavin (ug/g)12.7714.0010.4512.8014.820.760.49
Thiamine (ug/g)3.88 a12.08 b8.39 ab6.03 ab2.70 a1.170.04
Yolk
Glucose (mg/g)2.362.392.082.252.510.050.07
Grude protein (%)15.0214.2714.3415.042.510.180.23
Total soluble sugar (mg/g)2.70 a2.86 ab3.08 b2.61 a2.71 a0.050.014
Crude fat (%)29.6329.8329.9529.3731.370.290.20
Ash content (%)1.511.471.481.511.570.010.07
Moisture (%)52.9453.5353.3553.3451.050.360.15
Total cholesterol (mg/g)8.7010.599.8410.2511.390.440.42
Triglyceride (mg/g)26.0832.4029.1331.1732.420.910.10
Riboflavin (ug/g)11.13 bc12.31 c10.44 bc8.98 ab7.51 a0.540.01
Thiamine (ug/g)3.806.174.685.762.690.490.11
a–c Data within the same row marked with different small letters above the data indicate statistically significant differences (p < 0.05), while the same or no small letters indicate non-significant differences (p > 0.05).
Table 6. Effects of oregano oil on mineral elements of egg.
Table 6. Effects of oregano oil on mineral elements of egg.
ItemsConcentration (mg/kg)SEMp Value
0255075100
Egg white
Potassium (mg/kg)0.00520.00500.00620.00440.00540.020.11
Calcium (mg/kg)129.41198.56209.63193.91274.8026.270.60
Total phosphorus (mg/g)0.08 a0.08 ab0.11 b0.12 b0.13 c0.010.04
Magnesium (mg/g)0.170.210.250.190.250.010.31
Sodium (mg/g)1.882.112.161.932.190.080.72
Iron (mg/kg)41.4446.2034.7329.3274.686.550.21
Selenium (ug/kg)30.9729.6736.8833.0744.872.620.39
Zinc (mg/kg)1.290.711.210.511.390.160.36
Yolk
Potassium (mg/kg)0.00300.00360.00400.00340.00390.010.19
Calcium (mg/kg)1423.161553.552032.611558.431539.27130.180.68
Total phosphorus (mg/g)4.554.774.794.795.500.160.45
Magnesium (mg/g)0.270.310.410.290.280.030.50
Sodium (mg/g)0.600.700.710.750.660.030.56
Iron (mg/kg)194.49222.00240.99170.14166.0112.080.21
Selenium (ug/kg)150.87 ab387.19 c348.77 c228.43 bc39.72 a39.260.00
Zinc (mg/kg)44.5147.5446.1247.7952.151.690.74
a–c Data within the same row marked with different small letters above the data indicate statistically significant differences (p < 0.05), while the same or no small letters indicate non-significant differences (p > 0.05).
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MDPI and ACS Style

Xian, L.; Wang, Y.; Peng, D.; Zang, L.; Xu, Y.; Wu, Y.; Li, J.; Feng, J. Dietary Oregano Oil Supplementation Improved Egg Quality by Altering Cecal Microbiota Function in Laying Hens. Animals 2024, 14, 3235. https://doi.org/10.3390/ani14223235

AMA Style

Xian L, Wang Y, Peng D, Zang L, Xu Y, Wu Y, Li J, Feng J. Dietary Oregano Oil Supplementation Improved Egg Quality by Altering Cecal Microbiota Function in Laying Hens. Animals. 2024; 14(22):3235. https://doi.org/10.3390/ani14223235

Chicago/Turabian Style

Xian, Lili, Yan Wang, Da Peng, Lei Zang, Yidan Xu, Yuanyuan Wu, Jingjing Li, and Jing Feng. 2024. "Dietary Oregano Oil Supplementation Improved Egg Quality by Altering Cecal Microbiota Function in Laying Hens" Animals 14, no. 22: 3235. https://doi.org/10.3390/ani14223235

APA Style

Xian, L., Wang, Y., Peng, D., Zang, L., Xu, Y., Wu, Y., Li, J., & Feng, J. (2024). Dietary Oregano Oil Supplementation Improved Egg Quality by Altering Cecal Microbiota Function in Laying Hens. Animals, 14(22), 3235. https://doi.org/10.3390/ani14223235

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