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Article

Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum

by
Miguel Rabelo-Ruiz
1,
Juan José Ariza-Romero
2,
María Jesús Zurita-González
2,3,
Antonio Manuel Martín-Platero
1,
Alberto Baños
2,
Mercedes Maqueda
1,
Eva Valdivia
1,4,
Manuel Martínez-Bueno
1,4 and
Juan Manuel Peralta-Sánchez
1,*
1
Departamento de Microbiología, Universidad de Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain
2
Departamento de Microbiología y Biotecnología, DMC Research Center, Camino de Jayena s/n, 18620 Granada, Spain
3
Área de Endocrinología Molecular y Celular, Fundación Instituto de Investigación Sanotaria de Santiago de Compostela (FIDIS), Complejo Hospitalario Universitario De Santiago (CHUS), Servicio Gallego de Salud (SERGAS), 15706 Santiago de Compostela, Spain
4
Instituto de Biotecnología, Universidad de Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain
*
Author to whom correspondence should be addressed.
Animals 2021, 11(2), 448; https://doi.org/10.3390/ani11020448
Submission received: 7 January 2021 / Revised: 21 January 2021 / Accepted: 5 February 2021 / Published: 9 February 2021
(This article belongs to the Special Issue Antibiotic Alternatives in Animal Production)
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Abstract

:

Simple Summary

The misuse of antibiotics has led several countries to ban their use as prophylactics against bacterial diseases or as growth promoters in livestock and poultry. Phytobiotics (bioactive compounds extracted from plants) are one of the alternatives, due to their antimicrobial activity and its modulation of the gut microbiota and the improvement of productive properties. Garlic and onion extracts, rich in antimicrobial compounds, are of the most promising alternative to antibiotics. We supplemented a garlic- and onion-based product in the diet to laying hens at the beginning of their productive life. The group supplied with this product produced in one month more eggs and with bigger size. This increase in production was accompanied by changes in the bacterial community of the gut. These changes in the microbiota suggest an improvement in food digestibility, as the most important changes produced by these compounds occur in the most distal parts of the gut. The relative abundance of beneficial Lactococcus in the ileum and Lactobacillus in the cecum increased in the experimental group. Both genera are known to have beneficial effects on host. These results are very promising for the use of these compounds in poultry for short periods.

Abstract

Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on the hosts. These results are very promising for the use of these compounds in poultry for short periods.

1. Introduction

The abusive and inappropriate use of antibiotics in the animal production industry and clinical medicine has favored the selection of resistant bacteria and the spread of antibiotic resistance worldwide [1,2,3]. As a consequence, numerous countries have banned the use of most antibiotics as growth-promoters in livestock and poultry [4,5,6,7,8]. Some works predicted that these policies will increase production costs and final product prices [9,10], so the animal production industry is searching for efficient alternatives to the use of antibiotics as growth promoters in livestock, poultry and aquaculture [11,12,13]. Bacteriocins, bacteriophages, phytobiotics, probiotics, prebiotics and synbiotics have been proposed as the most promising alternatives [14,15].
Phytobiotics are bioactive compounds supplemented in the diet to improve the health and performance of farm animals [16,17]. Like antibiotics, phytobiotics can directly affect pathogenic bacteria, acting as antimicrobials [16,18] or by blocking some membrane receptors in pathogenic bacteria, making their adhesion to the intestinal mucosa difficult (reviewed in [16]). Phytobiotics can also act as prebiotics, supplying specific substrates and stimulating the growth of beneficial bacteria, or acting as growth-promoter metabolites [13,16]. Interestingly, phytobiotics may modulate the microbiota-gut-immune system, especially thorough antioxidant and anti-inflammation activities of these compounds [19]. Moreover, phytobiotics increase digestive enzyme activity, enhance feed conversion and hence improve the productive parameters of farm animals [17,19]. These improvements in digestive function have been related to the growth of beneficial bacteria in the cecum of broilers supplemented with phytobiotics, especially lactic acid bacteria such as lactobacilli and bifidobacteria [20,21]. These bacterial groups improve the host’s health by interacting with and training the immune system, allowing the host to allocate resources to production traits [22,23].
Extracts from plants of the Alliaceae family, mainly garlic (Allium sativum), onion (Allium cepa) and leek (Allium porrum) produce a wide variety of compounds showing antimicrobial activity, known of since ancient times [24,25,26]. The supplementation of these compounds has shown promising results in the health and productive parameters of several farm animals such as sheep [27], goats [28], cattle [29], pigs [30], broilers [31,32] and fishes [33]. These compounds improve intestinal functions such as rumen fermentations or energy-related blood metabolites, contributing to animal health and productivity [27,34]. In poultry, allium extracts supplemented in diets produce significant modulatory effects on growth, performance indices, lipid metabolism, gut ecosystem as well as immune responses, especially when poultry are experiencing stress and disease challenge conditions [35]. Most of these compounds are secondary metabolites, volatile organosulfur compounds, mainly thiosulfites and thiosulfonates which are responsible for the pungent odor, such as propyl thiosulfinate (PTS) and propyl propane thiosulfonate (PTSO) [24,36,37]. In in vitro experiments, these compounds showed anti-inflammatory properties in alveolar macrophages from pig lumps [36], antimicrobial activity against bacterial strains isolated from pig feces [37] and against Gram-negative and Gram-positive multidrug-resistant bacteria isolated from human fecal samples [38].
Previous works showed that experimental supplementation of garlic in the diet did not result in an increase in egg production [39,40,41,42], or even in decrease in egg productivity [43]. However, in a recent paper, Abad et al. [44] showed that a commercial Allium compound has positive effects on egg production. These differences in productivity effects due to Allium additives may be due to dose, duration of feeding or processing techniques [45]. In this manuscript, we hypothesize that the supplementation of Allium compounds in the diet of laying hens will affect bacterial community composition as well as egg production. We predict that a commercial Alliaceae extract supplemented in the diet of laying hens, similar to that used by Abad et al. [44] and based on PTS and PTSO, produces beneficial shifts in the gut microbiota and increase productive parameters, i.e., the number of laid eggs and their weights.

2. Materials and Methods

2.1. Experimental Animals and Facilities

The experiment was performed in 2014 at an experimental farm (Granja Avícola Gil, SL, Alhendín, Granada, Spain). Laying hens (Hy Line Brown) were placed in cages at the age of 16 weeks with food and water ad libitum and kept at 20 °C ± 2 °C and 78% ± 3% relative humidity (average ± standard deviation), under a photoperiod of 16 h per day throughout the experimental procedure.

2.2. Experimental Design and Sampling Collection

One hundred and eighty experimental laying hens were housed in groups of 6 hens per cage, with treatment groups being randomly distributed between production lines. Control hens (90 hens, 15 cages, 6 animals per cage) received a basal fodder diet (Supplementary Table S1), while experimental hens (90 hens, 15 cages, 6 animals per cage) received the same diet supplemented in feed with a commercial Alliaceae extract, Garlicon40 (DOMCA SAU, Granada, Spain) at a final concentration of 150 mg/kg (60 mg of PTSO per kg of feed).
The acclimation period lasted 15 days and then daily egg production (number of eggs) and their weight were recorded every working day (15 days of sampling). On day 30 after experiment started, 5 control and 8 experimental hens selected at random from different cages were euthanized by an intrathoracic injection of 2 mL/hen of T-61 (Intervet, Salamanca, Spain). Immediately after being slaughtered, the hens were dissected and the ileum and cecum were collected using sterile material. Each portion was homogenized in buffered peptone broth and aliquoted with 10% sucrose and finally frozen at −80 °C. Afterwards, the aliquots were lyophilized (LyoQuest, TELSTAR Technologies SL, Barcelona, Spain). No animal died during the experimental period due to illness or malnutrition.

2.3. High-throughput Sequencing

A total of 20 mg of lyophilized samples were used for bacterial DNA extraction from ileum and cecum samples using the QIAamp DNA Stool Mini Kit (QIAGEN). Amplicon libraries of the V4 region of the 16S rRNA gene were produced from total bacterial DNA by PCR amplification using primer pair 515f (5′-GTGCCAGCMGCCGCGGTAA-3′) and 786r (5´-GGACTACHVGGGTWTCTAAT-3′) with barcodes on the forward primer following Illumina library preparation (see Supplementary Materials and library preparation details in [46]). High-throughput sequencing was performed on Illumina MiSeq platform in the Scientific Instrumental Center at the University of Granada (Spain).
Subsequent analyses were performed with QIIME2 v2018.02 [47]. Primer trimming and pair joining were performed using default parameters. Afterwards, quality-filtering and sequence clustering were carried out using the Deblur algorithm, a sub-operational-taxonomic-unit approach that removes low quality sequences as well as sequencing errors [48], with a sequence length trimming limit of 252 base pairs. This algorithm allows an Amplicon Sequence Variant (ASV) table to be created. The fragment insertion script implemented in QIIME2 was used to perform a sequence alignment and construct a de-novo phylogenetic tree [49]. Taxonomy assignment was based on Greengenes 13_08 with a similarity of 99% [50]. Finally, non-bacterial sequences, i.e., chloroplasts and mitochondria, were removed from the sub-OTU table, although Cyanobacteria were retained in subsequent analyses [51].

2.4. Statistics

We used General Linear Models (GLM) to explore the effect of the treatment, sampling date and their interaction in number of eggs produced per day, mean egg size per day and alpha diversity indexes. For bacterial diversity analyses, we rarefied the ASV table at 2500 sequence depth per sample. We calculated different alpha diversity within sample diversity [52], indexes from the ASV table: bacterial OTU richness (or number of observed OTUs), evenness [53], Faith’s phylogenetic diversity index [54] and Shannon diversity index [55]. Residuals of the number of eggs, mean egg size and alpha diversity indexes after the analyses followed a normal distribution (Kolmogorov-Smirnov normality test; p > 0.20), validating the use of parametric statistical tests. These analyses were performed using STATISTICA 12.5 (Statsoft, Tulsa, OK, USA).
Difference in genera and phyla relative abundance between control and treated hens were explored by means of Linear Discriminant Analysis (LDA) Effect Size (LEfSe) [56]. LEfSe analyses were performed on the Galaxy web platform, run from a public server [57].
Weighted and unweighted UniFrac distance [58,59] were used to calculate beta diversity distance matrixes [differences between sample diversity, 52]. While weighted UniFrac gives more importance to the most abundant bacteria, unweighted UniFrac gives more importance to rare bacteria in the sub-OTUs as it takes into account their presence or absence regardless of abundance [59]. PERMANOVA was performed in order to test these effects on both UniFrac distance matrixes using PRIMER-7 software. Principal Coordinate Analyses were performed in order to visualize the first two PCoA axes using Emperor 2018.2.0 [60].

3. Results

3.1. Effect of Allium Supplementation on Egg Productivity of Laying Hens

Laying hens supplemented with the Alliaceae extract had higher egg production than the control group (Table 1, Figure 1A). While the control group decreased production throughout the experimental period in just 30 days (average egg number (standard error): 47.07 (0.79), the experimental group increased the number of eggs produced (52.07 (0.79); see interaction term in Table 1, Figure 2A).
The size of the eggs was also significantly affected by our experimental manipulation. Laying hens supplemented with the Alliaceae extract laid larger eggs (70.16 (0.18)) than the control group (68.85 (0.18)) (Table 1; Figure 1B). During the experimental period, egg weight in experimental hens increased, while egg size slightly decreased in the control group (see interaction term in Table 1, Figure 2B).

3.2. Changes in Bacterial Community Composition

The gut microbiota of laying hens is dominated by Firmicutes, Bacteroidetes and Proteobacteria. The relative abundance of these phyla depended on the gut region and treatment group (Figure 3). Firmicutes dominated in the ileum while Proteobacteria is dominant in the cecum. It is noteworthy that some minority phyla such as Elusimicrobia or Synergistetes decreased in the cecum of laying hens supplemented with the Alliaceae extract (Figure 3). However, only phylum OP8 differed significantly between treatments in both the ileum and cecum (Figure 4).
At the genus level, Lactococcus (Firmicutes) and an unidentified genus of Anaeroplasmataceae (Tenericutes) increased in the ileum while Bulleidia (Firmicutes), Bacteroides (Bacteroidetes) and an unknown genus of the phyla OP8 decreased in the supplemented hens (Figure 4, Supplementary Figures S1 and S2). In the cecum, two unknown genera of α-Proteobacteria and Lactobacillus (Firmicutes) increased significantly in the supplemented group. Anaerobiospirillum and Acinetobacter from Gammaproteobacteria, the genus RFN20 (Firmicutes), an unknown genus from Bacteroidetes and an unknown genus of OP8 decreased significantly in laying hens supplemented with the Alliaceae extract (Figure 4, Supplementary Figures S1 and S3).

3.3. Effect of Allium Compound Supplementation on Alpha and Beta Diversity

Supplementing the diet of laying hens with the Alliaceae extract did not affect any of the alpha diversity indexes after 30 days of treatment in any of the gut regions, ileum or cecum (Table 2).
After 30 days of treatment, the bacterial community in laying hens varied significantly between the control and the supplemented hens (Table 3), in both the ileum and cecum samples, forming clear non-overlapping clusters (Figure 5). Interestingly, changes in the bacterial community between the ileum and cecum were similar between control and supplemented hens (see interaction terms in Table 3). Within each gut region, samples from the same treatment level clustered significantly together (Table 3, Figure 5), except for weighted UniFrac metrics in the ileum, showing a marginally significant trend (Table 3). Our experiment affected both, abundant and rare bacterial taxa, as it shows weighted and unweighted UniFrac results, respectively (Table 3, Figure 5).

4. Discussion

This study found that laying hens experimentally supplemented with Allium by-product compounds, based mainly on PTS and PTSO, significantly increased the number of laid eggs, as well as their size, after only 30 days of treatment. These productive increases in egg production and quality were accompanied by shifts in ileum and cecum microbiota, where some bacterial groups differed between the supplemented and the control group.
Phytobiotics have shown promising results in the health, performance and productivity of laying hens. Diet supplementation with Allium compounds possess beneficial effects: significantly reduces cholesterol levels in the plasma of laying hens [61,62,63,64], in egg contents [63] and even protects against several diseases, including cancer [65]. Supplementation with different phytobiotics such as black cumin seeds or leaves and extracts from the Lamiaceae family (such as peppermint, sage, rosemary, thyme and oregano) increase egg production and egg weight [64,66,67,68,69,70]. However, results for egg production in laying hens with their diet supplemented by Allium compounds are contradictory. Some studies pointed out the lack of effect on egg production or egg weight when hens were provided with different garlic preparations such as garlic paste, oil or powder [62,63,71]. However, and in accordance with our results, supplementation of garlic powder shows an increase in egg production [63]. Olobatoke and Mulugeta [72] found an increase in egg weight and a reduction in laying rate, but in laying hens supplemented with high doses of garlic powder. The differences in the associations between garlic-based compounds supplementation and these variables may be related with breeds of hen and the preparation and presentation of the garlic products [62,63,71], probably related to the composition and quantity of sulfur components [63].
Garlic, onion and its relatives are plants rich in several volatile organosulfur compounds responsible for the pungent odor and antimicrobial properties [24,71]. Allicin was one of the first compounds with antimicrobial activity to be isolated from garlic [73], although its instability does not allow it to be used in livestock and poultry [24]. Allicin has been substituted by other compounds in the use of Allium-derived substances in animal production and welfare, such as PTS and PTSO, by-products of the initial compounds present in garlic and onion such as alliin and propiin [37,74]. PTS is quite instable but it converts rapidly into PTSO, a more stable compound [37]. PTS and PTSO preparations have been shown to increase propionate concentrations in lamb rumen, improve weight gain and reduce non-esterified fatty acids and β-hydroxybutyrate [27]. Interestingly, high concentrations of garlic powder (5%) shows stronger garlic flavor in the eggs compared with those eggs laid by control and laying hens supplemented with lower doses (3%) [72]. In spite of this negative effects in organoleptic properties of eggs, PTSO did not seem to alter animal derived products. For instance, milk maintains its organoleptic properties after two months of PTSO supplementation in the diet of cows [29].
In broilers, supplementation of PTS and PTSO in the diet produces changes in the morphology and histology of the ileum and increases mucosa complexity in the gut [75]. It also produces shifts in the proximal intestinal microbiota of broilers, maintaining mucosal enzyme activity but improving food digestibility [76], with an associated increase in body weight [75]. These compounds also reduce Salmonella abundance in the ileum and Escherichia coli in the cecum of broilers [75]. Besides this direct effect of Allium-derived compounds, bacterial communities of the gut exclude pathogenic bacteria and enhance the development of the intestinal mucosa, epithelium and lamina propria, resulting in an improvement in farm animals’ health [77]. Reducing pathogenic bacteria brings relief to intestinal challenge and immune stress and hence the host can allocate resources to other traits [78,79]. In this sense, our results showed a significant shift in the bacterial community in the ileum and cecum in laying hens supplemented with Allium-derived compounds, as shown by the UniFrac analyses. Our results agree with previous findings where these changes in microbiota are especially important in the most distal parts of the gut of monogastric animals, such as the cecum [37]. The underlying action mechanisms of phytobiotics have not been explored yet, so it would deserve further studies, especially those related with changes in gut mucosa, the immune system and food digestibility.
Our results show that the relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in laying hens supplemented with PTS and PTSO, while egg production improved. A recent paper based in culture-dependent techniques showed similar increase in egg production and fecal Lactobacillus counts in laying hens, supplemented with even lower doses of PTSO than the present work [44]. The increase in relative and absolute bacterial abundances of both Lactococcus and Lactobacillus produce beneficial effects in poultry and farm animals [80]. Han et al. [81] found that the relative abundance of Lactococcus in the cecum of broilers was positively correlated with body weight. Moreover, supplementation with a phytobiotic in laying hens increased Lactobacillus relative abundance in the cecum and simultaneously improved egg production as well as egg weights [68]. Most lactic acid bacteria have an intimate relationships with the health of their animal hosts [82], so these strains have been widely used as probiotics due to their many beneficial properties [83,84,85]. In this sense, these bacteria reduce the intestinal pH by producing lactic acid, and hence, inhibiting the proliferation of pathogenic bacteria (revised in [86]). The action of Lactobacillus may be also related with the reduction in the adhesion ability of Salmonella or of pathogenic bacteria as some strains of Clostridium spp. or E. coli [80]. Moreover, the levels of Lactobacillus could play a major role in promoting and maintaining intestinal inflammation, especially during inflammatory disease [80]. In this sense, lactic acid bacteria also increase the histological complexity of the gut and stimulate the immune response of the mucosa [86,87]. Despite not exploring the variables involved in these effects, a net positive effect in both lactic acid bacteria and egg production was found.
In animals, Bacteroidetes is present in the small and large intestine, although its relative abundance is much higher in the latter [82]. Our experimental procedure produced a significant reduction in the relative abundance of Bacteroides in the ileum and in an unidentified genus of the Order Bacteroidales in the cecum. Bacteroidetes species are involved in several metabolic activities in the gut, from carbohydrate fermentation to bile acid degradation (Bry et al., 1996; Phillips, 2009). Interestingly, Peinado et al. [88] found using qPCR that the absolute abundance of Bacteroides in Cobb broiler guts negatively correlated with Lactobacillus populations. In that study, they found an increase in Bacteroides and broiler performance in animals supplemented with PTSO while Lactobacillus abundance decreased. We can only speculate that these discrepancies may be due to the use of different molecular techniques (qPCR vs. high-throughput sequencing, absolute vs. relative abundances), different hen breeds (Cobb vs. Hy Line Brown) and more importantly, differences in age and sex. For instance, ileum microbiota differs significantly between male and female broilers only 3 days after hatching [89]. Moreover, age, sex and breed has a strong effect on bacterial community in the gut of broilers [90]. Further research is needed to clarify the effects of these confounding factors and explore new possibilities in broilers and laying hens as shown by the depletion of Bacteroides observed in obese children [91].
Our supplementation with PTS and PTSO depleted other genera that may cause negative effects on their hosts. Acinetobacter (Moraxellaceae, Gammaproteobacteria) is a common bacterium in soil environments and related with infections in immune-depressed patients [92]. Anaerobiospirillum (Succinivibrionaceae, Gammaproteobacteria) is a strict anaerobic genus causing septicemia and diarrhea in humans [93]. Some other taxa are poorly described so the effect of the reduction in abundance is unknown. These taxa include members of the Erysipelotrichaceae family, such as Bulleida and the genus RFN20, and an unidentified genus in the candidate phylum OP8. In this sense, Aminicenantes, the proposed name for this phylum, is poorly characterized and the few described strains cover a wide range of environments [94]. Similarly, other groups increased their abundance, such as a genus of Anaeroplasmataceae, anaerobic obligate commensals in the rumen of some mammals, the role of which has not yet been properly described [95] or the genus belonging to the Order RF32, the abundance of which correlates with histopathology and colonic inflammation in ray challenge with E. coli. Due to the lack of available information about the ecology and function of these groups in the gut, we cannot explain the significance of these changes in abundance in most of these strains in the treated hens. Culture-based methods are experiencing a rebirth in order to fill the gap in the knowledge of the huge amount of new microorganisms and diversity that next-generation technology is uncovering [96].

5. Conclusions

Our experimental supplementation of PTS and PTSO compounds in diet of laying hens increased their egg production and size, while producing shifts in the bacterial communities in the ileum and cecum of the hens. These results are very promising for the use of these phytobiotics in poultry for short periods (4 weeks in this study). Future research is necessary to understand the underlying mechanisms involved in these improvements, regarding the immune system, food digestibility and for longer exposition periods.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2615/11/2/448/s1; Figure S1. Bar plot of the relative bacterial abundance at the genus level in different gut regions of laying hens and treatments. Control refers to laying hens fed a basal diet while Garlicon refers to experimental laying hens fed a basal diet supplemented with the commercial Alliaceae extract. The sixteen most abundance genera are shown in a unique color set. The color of the rest of genera (less abundant) are repeated every 8 colors; Figure S2. Linear Discriminant Analysis Effect Size (Lefse) showing genera from the ileum that significantly differ between control and experimentally supplemented with a commercial Alliaceae laying hens. Bars showed relative abundance of the genus in each sample. Solid line represents mean relative abundance while dashed line represent the median. k: kingdom; p: phylum; c: class; o: order; f: family; and g: genus; Figure S3. Linear Discriminant Analysis Effect Size (Lefse) showing genera from the cecum (ciego) that significantly differ between control and experimentally supplemented with a commercial Alliaceae laying hens. Bars showed relative abundance of the genus in each sample. Solid line denotes relative abundance while dashed line showed median relative abundance. k: kingdom; p: phylum; c: class; o: order; f: family; and g: genus; Table S1: Nutritional information of the basal feed employed in laying hens.

Author Contributions

Conceptualization, J.J.A.-R., M.J.Z.-G., A.B. and M.M.-B.; methodology, M.R.-R., J.J.A.-R., M.J.Z.-G., A.M.M.-P., A.B., M.M., E.V., M.M.-B. and J.M.P.-S.; validation, A.B., M.M.-B. and J.M.P.-S.; formal analysis, M.R.-R., J.J.A.-R., M.J.Z.-G. and J.M.P.-S.; resources, A.B. and M.M.-B.; data curation, M.R.-R. and J.M.P.-S.; writing—original draft preparation, J.M.P.-S.; writing—review and editing, M.R.-R., J.J.A.-R., M.J.Z.-G., A.M.M.-P., A.B., M.M., E.V., M.M.-B. and J.M.P.-S.; supervision, A.B., M.M.-B. and J.M.P.-S.; project administration, A.B. and M.M.-B.; funding acquisition, A.B. and M.M.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Spanish Ministry of Science and Innovation, Consejería de Economía, Innovación, Ciencia y Empleo (Junta de Andalucía), the University of Granada-CEI BioTic (Project No. P-BS-37), and the INTERCONECTA program (Spanish Ministry of Economy and Competitiveness).

Institutional Review Board Statement

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, the European directive for the protection of animal welfare in research (Directive 2010/63/EU) and the ‘Guide for the Care and Use of Laboratory Animals’, as promulgated by the National Institute of Health (Ministerio de Sanidad, Consumo y Bienestar Social) following Spanish legislation (Real Decreto 53/2013). Protocols and procedures were approved by the Ethics Committee of Laboratory Animals of the University of Granada (Spain) (Ref. No. CEEA-2018-227).

Data Availability Statement

Sequences are available in the Sequence Read Archive (SRA) in the GenBank-NCBI webpage1 under Accession Nos. SAMN09603288 to SAMN9603307 and SAMN09603326 to SAMN 09603341.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Wise, R. The worldwide threat of antimicrobial resistance. Curr. Sci. 2008, 95, 181–187. [Google Scholar]
  2. World Health Organization. Antimicrobial Resistance. 2018. Available online: http://www.who.int/antimicrobial-resistance/en/ (accessed on 17 May 2018).
  3. Levy, S.B.; Marshall, B. Antibacterial resistance worldwide: Causes, challenges and responses. Nat. Med. 2004, 10, S122–S129. [Google Scholar] [CrossRef]
  4. European Commission. Regulation (EU) 2019/4 of the European Parliament and of the Council of 11 December 2018 on the manufacture, placing on the market and use of medicated feed, amending Regulation (EC) No 183/2005 of the European Parliament and of the Council and repealing Council Directive 90/167/EEC (Text with EEA relevance). Off. J. Eur. Union 2018, 62, L:2019:004:TOC. [Google Scholar]
  5. Wierup, M. The Swedish experience of the 1986 year ban of antimicrobial growth promoters, with special reference to animal health, disease prevention, productivity, and usage of antimicrobials. Microb. Drug. Resist. 2001, 7, 183–190. [Google Scholar] [CrossRef] [Green Version]
  6. Bengtsson, B.; Wierup, M. Antimicrobial resistance in Scandinavia after a ban of antimicrobial growth promoters. Anim. Biotechnol. 2006, 17, 147–156. [Google Scholar] [CrossRef]
  7. Food and Drug Administration. New Animal Drugs and New Animal Drug Combination Products Administered in or on Medicated Feed or Drinking Water of Food-Producing Animals: Recommendations for Drug Sponsors for Voluntarily Aligning Product Use Conditions With GFI #209; Food and Drug Administration: Silver Spring, MA, USA, 2013.
  8. European Commission. Commision Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. Off. J. Eur. Union 2003, 40, 29–43. [Google Scholar]
  9. Hardy, B. The issue of antibiotic use in the livestock industry. What have we learned? Anim. Biotechnol. 2002, 13, 129–147. [Google Scholar] [CrossRef] [PubMed]
  10. Hayes, D.J.; Jensen, H.H.; Backstrom, L.; Fabiosa, J. Economic impact of a ban on the use of over the counter antibiotics in US swine rations. Int. Food Agribus. Man. 2001, 4, 81–97. [Google Scholar] [CrossRef]
  11. Prasanta, J.; Sutanu, K.; Utsa, R.; Mritunjoy, P.; Ashutosh, B.; Singhand, K.; Krishna, K. Phytobiotics in aquaculture health management: A review. J. Entomol. Zool. 2018, 6, 1422–1429. [Google Scholar]
  12. Ferket, P.R. Alternatives to antibiotics in poultry production: Responses, practical experience and recommendations. In Nutritional Biotechnology in the Feed and Food Industries; Lyons, T.P., Jacques, K.A., Eds.; Nottinghan University Press: Nottinghan, UK, 2004; pp. 57–67. [Google Scholar]
  13. Allen, H.K.; Levine, U.Y.; Looft, T.; Bandrick, M.; Casey, T.A. Treatment, promotion, commotion: Antibiotic alternatives in food-producing animals. Trends Microbiol. 2013, 21, 114–119. [Google Scholar] [CrossRef] [Green Version]
  14. Joerger, R.D. Alternatives to antibiotics: Bacteriocins, antimicrobial peptides and bacteriophages. Poult. Sci. 2003, 82, 640–647. [Google Scholar] [CrossRef]
  15. Hume, M.E. Historical perspective: Prebiotics, probiotics, and other alternatives to antibiotics. J. Dairy Sci. 2010, 93, 191–192. [Google Scholar]
  16. Vidanarachchi, J.K.; Mikkelsen, L.L.; Sims, I.; Iji, P.A.; Choct, M. Phytobiotics: Alternatives to antibiotic growth promoters in monogastric animal feeds. Recent Adv. Anim. Nutr. 2005, 15, 131–144. [Google Scholar]
  17. Windisch, W.; Schedle, K.; Plitzner, C.; Kroismayr, A. Use of phytogenic products as feed additives for swine and poultry. J. Anim. Sci. 2008, 86, E140–E148. [Google Scholar] [CrossRef]
  18. Abd El-Ghany, W.A.; Ismail, M. Tackling experimental colisepticaemia in broiler chickens using phytobiotic essential oils and antibiotic alone or in combination. Iran. J. Vet. Res. 2014, 15, 110–115. [Google Scholar]
  19. Gheisar, M.M.; Kim, I.H. Phytobiotics in poultry and swine nutrition—A review. Ital. J. Anim. Sci. 2018, 17, 92–99. [Google Scholar] [CrossRef]
  20. Attia, G.; El-Eraky, W.; Hassanein, E.; El-Gamal, M.; Farahat, M.; Hernandez-Santana, A. Effect of dietary inclusion of a plant extract blend on broiler growth performance, nutrient digestibility, caecal microflora and intestinal histomorphology. Int. J. Poult. Sci. 2017, 16, 344–353. [Google Scholar] [CrossRef] [Green Version]
  21. Guo, F.C.; Williams, B.A.; Kwakkel, R.P.; Li, H.S.; Li, X.P.; Luo, J.Y.; Li, W.K.; Verstegen, M.W.A. Effects of mushroom and herb polysaccharides, as alternatives for an antibiotic, on the cecal microbial ecosystem in broiler chickens. Poult. Sci. 2004, 83, 175–182. [Google Scholar] [CrossRef]
  22. Al-Yasiry, A.R.M.; Kiczorowska, B.; Samolinska, W.; Kowalczuk-Vasilev, E.; Kowalczyk-Pecka, D. The effect of Boswellia serrata resin diet supplementation on production, hematological, biochemical and immunological parameters in broiler chickens. Animal 2017, 11, 1890–1898. [Google Scholar] [CrossRef] [Green Version]
  23. Liu, Y.; Song, M.; Che, T.M.; Almeida, J.A.S.; Lee, J.J.; Bravo, D.; Maddox, C.W.; Pettigrew, J.E. Dietary plant extracts alleviate diarrhea and alter immune responses of weaned pigs experimentally infected with a pathogenic Escherichia coli. J. Anim. Sci. 2013, 91, 5294–5306. [Google Scholar] [CrossRef] [Green Version]
  24. Amagase, H.; Petesch, B.L.; Matsuura, H.; Kasuga, S.; Itakura, Y. Intake of garlic and its bioactive components. J. Nutr. 2001, 131, 955S–962S. [Google Scholar] [CrossRef] [PubMed]
  25. Ye, C.-L.; Dai, D.-H.; Hu, W.-L. Antimicrobial and antioxidant activities of the essential oil from onion (Allium cepa L.). Food Control. 2013, 30, 48–53. [Google Scholar] [CrossRef]
  26. Harris, J.C.; Cottrell, S.L.; Plummer, S.; Lloyd, D. Antimicrobial properties of Allium sativum (garlic). Appl. Microbiol. Biotechnol. 2001, 57, 282–286. [Google Scholar] [CrossRef] [PubMed]
  27. Anassori, E.; Dalir-Naghadeh, B.; Valizadeh-Keshmeshtappeh, M.; Jafari, S. A comparative study on the efficacy of Garlicon and monensin supplementation on blood metabolites and performance of fattening lambs. Livest. Sci. 2017, 199, 74–78. [Google Scholar] [CrossRef]
  28. Zhu, Z.; Hang, S.; Zhu, H.; Zhong, S.; Mao, S.; Zhu, W. Effects of garlic oil on milk fatty acid profile and lipogenesis-related gene expression in mammary gland of dairy goats. J. Sci. Food Agric. 2013, 93, 560–567. [Google Scholar] [CrossRef] [PubMed]
  29. Abad, P.; Arroyo-Manzanares, N.; Gil, L.; Garcia-Campana, A.M. Use of onion extract as a dairy cattle feed supplement: Monitoring propyl propane thiosulfonate as a marker of its effect on milk attributes. J. Agric. Food Chem. 2017, 65, 793–799. [Google Scholar] [CrossRef]
  30. Abad, P.; Arroyo-Manzanares, N.; Rivas-Montoya, E.; Ochando-Pulido, J.M.; Guillamon, E.; García-Campaña, A.M.; Martinez-Ferez, A. Effects of different vehiculization strategies for the Allium derivative propyl propane thiosulfonate during dynamic simulation of the pig gastrointestinal tract. Can. J. Anim. Sci. 2018, 99, 244–253. [Google Scholar] [CrossRef]
  31. Jimoh, A.; Olorede, B.; Abubakar, A.; Fabiyi, J.; Ibitoye, E.; Suleiman, N.; Garba, S. Lipids profile and haematological indices of broiler chickens fed garlic (Allium sativum)-supplemented diets. J. Vet. Adv. 2012, 2, 474–480. [Google Scholar]
  32. Lee, J.S.; Kim, M.J.; Park, S.H.; Lee, S.B.; Wang, T.; Jung, U.S.; Im, J.; Kim, E.J.; Lee, K.W.; Lee, H.G. Effects of dietary mixture of garlic (Allium sativum), coriander (Coriandrum sativum) and probiotics on immune responses and caecal counts in young laying hens. J. Anim. Physiol. Anim. Nutr. 2017, 101, e122–e132. [Google Scholar] [CrossRef]
  33. Natasya-Ain, R.; Eirna-Liza, N.; Jasmin, M.; Karim, M. Antibacterial activity of garlic extracts on fish pathogenic bacteria. J. Environ. Biol. 2018, 39, 808–812. [Google Scholar] [CrossRef]
  34. Busquet, M.; Calsamiglia, S.; Ferret, A.; Carro, M.D.; Kamel, C. Effect of garlic oil and four of its compounds on rumen microbial fermentation. J. Dairy Sci. 2005, 88, 4393–4404. [Google Scholar] [CrossRef] [Green Version]
  35. Kothari, D.; Lee, W.-D.; Niu, K.-M.; Kim, S.-K. The genus Allium as poultry feed additive: A review. Animals 2019, 9, 1032. [Google Scholar] [CrossRef] [Green Version]
  36. Liu, Y.; Song, M.; Che, T.M.; Bravo, D.; Pettigrew, J.E. Anti-inflammatory effects of several plant extracts on porcine alveolar macrophages in vitro. J. Anim. Sci. 2012, 90, 2774–2783. [Google Scholar] [CrossRef]
  37. Ruiz, R.; Garcia, M.P.; Lara, A.; Rubio, L.A. Garlic derivatives (PTS and PTS-O) differently affect the ecology of swine faecal microbiota in vitro. Vet. Microbiol. 2010, 144, 110–117. [Google Scholar] [CrossRef]
  38. Sorlozano-Puerto, A.; Albertuz-Crespo, M.; Lopez-Machado, I.; Jose Ariza-Romero, J.; Banos-Arjona, A.; Exposito-Ruiz, M.; Gutierrez-Fernandez, J. In vitro antibacterial activity of propyl-propane-thiosulfinate and propyl-propane-thiosulfonate derived from Allium spp. against Gram-negative and Gram-positive multidrug-resistant bacteria isolated from human samples. Biomed Res. Int. 2018, 2018, 7861207. [Google Scholar] [CrossRef] [Green Version]
  39. Mahmoud, K.Z.; Gharaibeh, S.M.; Zakaria, H.A.; Qatramiz, A.M. Garlic (Allium sativum) supplementation: Influence on egg production, quality, and yolk cholesterol level in layer hens. Asian Australas. J. Anim. Sci. 2010, 23, 1503–1509. [Google Scholar] [CrossRef]
  40. Ghasemi, R.; Zarei, M.; Torki, M. Adding medicinal herbs including garlic (Allium sativum) and thyme (Thymus vulgaris) to diet of laying hens and evaluating productive performance and egg quality characteristics. Am. J. Anim. Vet. Sci. 2010, 5, 151–154. [Google Scholar] [CrossRef]
  41. Asrat, M.; Zeryehun, T.; Amha, N.; Urge, M. Effects of supplementation of different levels of garlic (Allium sativum) on egg production, egg quality and hatchability of White Leghorn chicken. Livest. Res. Rural Dev. 2018, 30, 37. [Google Scholar]
  42. Omer, H.A.; Ahmed, S.M.; Abdel-Magid, S.S.; El-Mallah, G.M.; Bakr, A.A.; Fattah, M.M.A. Nutritional impact of inclusion of garlic (Allium sativum) and/or onion (Allium cepa L.) powder in laying hens’ diets on their performance, egg quality, and some blood constituents. Bull. Natl. Res. Cent. 2019, 43, 23. [Google Scholar] [CrossRef]
  43. Kaya, H.; Macit, M. Effect of inclusion of garlic (Allium sativum) powder at different levels and copper into diets of hens on performance, egg quality traits and yolk cholesterol content. Int. J. Poult. Sci. 2012, 11, 114–119. [Google Scholar] [CrossRef] [Green Version]
  44. Abad, P.; Arroyo-Manzanares, N.; Ariza, J.J.; Baños, A.; García-Campaña, A.M. Effect of Allium extract supplementation on egg quality, productivity, and intestinal microbiota of laying hens. Animals 2020, 2021, 41. [Google Scholar] [CrossRef] [PubMed]
  45. Khan, R.; Nikousefat, Z.; Tufarelli, V.; Naz, S.; Javdani, M.; Laudadio, V. Garlic (Allium sativum) supplementation in poultry diets: Effect on production and physiology. World Poult. Sci. J. 2012, 68, 417–424. [Google Scholar] [CrossRef]
  46. Peralta-Sanchez, J.M.; Martin-Platero, A.M.; Ariza-Romero, J.J.; Rabelo-Ruiz, M.; Zurita-Gonzalez, M.J.; Baños, A.; Rodriguez-Ruano, S.M.; Maqueda, M.; Valdivia, E.; Martinez-Bueno, M. Egg production in poultry farming is improved by probiotic bacteria. Front. Microbiol. 2019, 10, 1042. [Google Scholar] [CrossRef] [Green Version]
  47. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.; Asnicar, F.; et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 2019, 37, 852–857. [Google Scholar] [CrossRef]
  48. Amir, A.; McDonald, D.; Navas-Molina, J.A.; Kopylova, E.; Morton, J.T.; Xu, Z.Z.; Kightley, E.P.; Thompson, L.R.; Hyde, E.R.; Gonzalez, A.; et al. Deblur rapidly resolves single-nucleotide community sequence patterns. mSystems 2017, 2, e00191-16. [Google Scholar] [CrossRef] [Green Version]
  49. Janssen, S.; McDonald, D.; Gonzalez, A.; Navas-Molina, J.A.; Jiang, L.; Xu, Z.Z.; Winker, K.; Kado, D.M.; Orwoll, E.; Manary, M.; et al. Phylogenetic placement of exact amplicon sequences improves associations with clinical information. mSystems 2018, 3, e00021-18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E.L.; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 2006, 72, 5069–5072. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Ley, R.E.; Backhed, F.; Turnbaugh, P.; Lozupone, C.A.; Knight, R.D.; Gordon, J.I. Obesity alters gut microbial ecology. Proc. Natl. Acad. Sci. USA 2005, 102, 11070–11075. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Whitaker, R.H. Evolution and measurement of species diversity. Taxon 1972, 21, 213–251. [Google Scholar] [CrossRef] [Green Version]
  53. Pielou, E.C. Measurement of diversity in different types of biological collections. J. Theor. Biol. 1966, 13, 131. [Google Scholar] [CrossRef]
  54. Faith, D.P.; Baker, A.M. Phylogenetic diversity (PD) and biodiversity conservation: Some bioinformatics challenges. Evol. Bioinform. Online 2006, 2, 121–128. [Google Scholar] [CrossRef]
  55. Shannon, C.E. A mathematical theory of communication. Bell Syst. Tech. 1948, 27, 379–423. [Google Scholar] [CrossRef] [Green Version]
  56. Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W.S.; Huttenhower, C. Metagenomic biomarker discovery and explanation. Genome Biol. 2011, 12, R60. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W.S.; Huttenhower, C. LEfSe: Linear Discriminant Analysis Effect Size. 2011. Available online: https://huttenhower.sph.harvard.edu/galaxy/ (accessed on 8 February 2021).
  58. Lozupone, C.; Knight, R. UniFrac: A new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 2005, 71, 8228–8235. [Google Scholar] [CrossRef] [Green Version]
  59. Lozupone, C.A.; Hamady, M.; Kelley, S.T.; Knight, R. Quantitative and qualitative beta diversity measures lead to different insights into factors that structure microbial communities. Appl. Environ. Microbiol. 2007, 73, 1576–1585. [Google Scholar] [CrossRef] [Green Version]
  60. Vazquez-Baeza, Y.; Pirrung, M.; Gonzalez, A.; Knight, R. EMPeror: A tool for visualizing high-throughput microbial community data. Gigascience 2013, 2, 16. [Google Scholar] [CrossRef] [Green Version]
  61. Ao, X.; Yoo, J.S.; Lee, J.H.; Jang, H.D.; Wang, J.P.; Zhou, T.X.; Kim, I.H. Effects of fermented garlic powder on production performance, egg quality, blood profiles and fatty acids composition of egg yolk in laying hens. Asian Australas. J. Anim. Sci. 2010, 23, 786–791. [Google Scholar] [CrossRef]
  62. Chowdhury, S.; Smith, T. Effects of dietary garlic on cholesterol metabolism in laying hens. Poult. Sci. 2002, 81, 1856–1862. [Google Scholar] [CrossRef]
  63. Yalcin, S.; Onbasilar, E.E.; Reisli, Z.; Yalcin, S. Effect of garlic powder on the performance, egg traits and blood parameters of laying hens. J. Sci. Food Agric. 2006, 86, 1336–1339. [Google Scholar] [CrossRef]
  64. Abdel-Wareth, A.A.A.; Lohakare, J.D. Effect of dietary supplementation of peppermint on performance, egg quality, and serum metabolic profile of Hy-Line Brown hens during the late laying period. Anim. Feed Sci. Technol. 2014, 197, 114–120. [Google Scholar] [CrossRef]
  65. Omar, S.; Al-Wabel, N.J.S.P.J. Organosulfur compounds and possible mechanism of garlic in cancer. Saudi Pharm. J. 2010, 18, 51–58. [Google Scholar] [CrossRef] [Green Version]
  66. Radwan, N.; Hassan, R.; Qota, E.; Fayek, H. Effect of natural antioxidant on oxidative stability of eggs and productive and reproductive performance of laying hens. Int. J. Poult. Sci. 2008, 7, 134–150. [Google Scholar]
  67. Bölükbasi, S.C.; Erhan, M.K. Effect of dietary thyme (Thymus vulgaris) on laying hens performance and Escherichia coli (E. coli) concentration in feces. IJNES 2007, 1, 55–58. [Google Scholar]
  68. Park, J.H.; Song, T.H.; Kim, I. Egg production, egg quality, and cecal microbial populations of layers fed diets supplemented with fermented phytogenic feed additive. Turk. J. Vet. Anim. Sci. 2016, 40, 660–666. [Google Scholar] [CrossRef]
  69. Khan, S.H.; Anjum, M.A.; Parveen, A.; Khawaja, T.; Ashraf, N.M. Effects of black cumin seed (Nigella sativa L.) on performance and immune system in newly evolved crossbred laying hens. Vet. Q. 2013, 33, 15–21. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Bozkurt, M.; Kucukyilmaz, K.; Catli, A.U.; Cinar, M.; Bintas, E.; Coven, F. Performance, egg quality, and immune response of laying hens fed diets supplemented with mannan-oligosaccharide or an essential oil mixture under moderate and hot environmental conditions. Poult. Sci. 2012, 91, 1379–1386. [Google Scholar] [CrossRef]
  71. Lanzotti, V. The analysis of onion and garlic. J. Chromatogr. A 2006, 1112, 3–22. [Google Scholar] [CrossRef] [Green Version]
  72. Olobatoke, R.Y.; Mulugeta, S.D. Effect of dietary garlic powder on layer performance, fecal bacterial load, and egg quality. Poult. Sci. 2011, 90, 665–670. [Google Scholar] [CrossRef]
  73. Cavallito, C.J.; Bailey, J.H. Allicin, the antibacterial principle of Allium sativum I Isolation, physical properties and antibacterial action. J. Am. Chem. Soc. 1944, 66, 1950–1951. [Google Scholar] [CrossRef]
  74. Wettenhall, C. Allium: Ecology, Distribution and Cultivation; Nova Science Publishers Inc.: Hauppauge, NY, USA, 2020; 282p. [Google Scholar]
  75. Peinado, M.J.; Ruiz, R.; Echavarri, A.; Rubio, L.A. Garlic derivative propyl propane thiosulfonate is effective against broiler enteropathogens in vivo. Poult. Sci. 2012, 91, 2148–2157. [Google Scholar] [CrossRef] [PubMed]
  76. Peinado, M.J.; Ruiz, R.; Echavarri, A.; Aranda-Olmedo, I.; Rubio, L.A. Garlic derivative PTS-O modulates intestinal microbiota composition and improves digestibility in growing broiler chickens. Anim. Feed Sci. Technol. 2013, 181, 87–92. [Google Scholar] [CrossRef]
  77. Baurhoo, B.; Ferket, P.R.; Zhao, X. Effects of diets containing different concentrations of mannanoligosaccharide or antibiotics on growth performance, intestinal development, cecal and litter microbial populations, and carcass parameters of broilers. Poult. Sci. 2009, 88, 2262–2272. [Google Scholar] [CrossRef]
  78. Thomas, C.M.; Versalovic, J. Probiotics-host communication: Modulation of signaling pathways in the intestine. Gut Microb. 2010, 1, 148–163. [Google Scholar] [CrossRef]
  79. Gadde, U.; Kim, W.H.; Oh, S.T.; Lillehoj, H.S. Alternatives to antibiotics for maximizing growth performance and feed efficiency in poultry: A review. Anim. Health Res. Rev. 2017, 18, 26–45. [Google Scholar] [CrossRef]
  80. Gaggia, F.; Mattarelli, P.; Biavati, B. Probiotics and prebiotics in animal feeding for safe food production. Int. J. Food Microbiol. 2010, 141, S15–S28. [Google Scholar] [CrossRef]
  81. Han, G.G.; Kim, E.B.; Lee, J.; Lee, J.-Y.; Jin, G.; Park, J.; Huh, C.-S.; Kwon, I.-K.; Kil, D.Y.; Choi, Y.-J.; et al. Relationship between the microbiota in different sections of the gastrointestinal tract, and the body weight of broiler chickens. Springerplus 2016, 5, 911. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Donaldson, G.P.; Lee, S.M.; Mazmanian, S.K. Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 2016, 14, 20–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Song, A.A.-L.; In, L.L.A.; Lim, S.H.E.; Rahim, R.A. A review on Lactococcus lactis: From food to factory. Microb. Cell. Fact. 2017, 16, 55. [Google Scholar] [CrossRef] [Green Version]
  84. Zhang, Z.W.; Lv, J.L.; Pan, L.; Zhang, Y.G. Roles and applications of probiotic Lactobacillus strains. Appl. Microbiol. Biotechnol. 2018, 102, 8135–8143. [Google Scholar] [CrossRef] [PubMed]
  85. Picard, C.; Fioramonti, J.; Francois, A.; Robinson, T.; Neant, F.; Matuchansky, C. Review article: Bifidobacteria as probiotic agents—Physiological effects and clinical benefits. Aliment. Pharmacol. Ther. 2005, 22, 495–512. [Google Scholar] [CrossRef]
  86. Kiczorowska, B.; Samolinska, W.; Al-Yasiry, A.R.M.; Kiczorowski, P.; Winiarska-Mieczan, A. The natural feed additives as immunostimulants in monogastric animal nutrition—A review. Ann. Anim. Sci. 2017, 17, 605–625. [Google Scholar] [CrossRef] [Green Version]
  87. Baurhoo, B.; Letellier, A.; Zhao, X.; Ruiz-Feria, C.A. Cecal populations of lactobacilli and bifidobacteria and Escherichia coli populations after in vivo Escherichia coli challenge in birds fed diets with purified lignin or mannanoligosaccharides. Poult. Sci. 2007, 86, 2509–2516. [Google Scholar] [CrossRef]
  88. Peinado, M.J.; Echavarri, A.; Ruiz, R.; Suarez-Pereira, E.; Ortiz Mellet, C.; Garcia Fernandez, J.M.; Rubio, L.A. Effects of inulin and di-D-fructose dianhydride-enriched caramels on intestinal microbiota composition and performance of broiler chickens. Animal 2013, 7, 1779–1788. [Google Scholar] [CrossRef] [Green Version]
  89. Lumpkins, B.S.; Batal, A.B.; Lee, M. The effect of gender on the bacterial community in the gastrointestinal tract of broilers. Poult. Sci. 2008, 87, 964–967. [Google Scholar] [CrossRef]
  90. Kers, J.G.; Velkers, F.C.; Fischer, E.A.J.; Hermes, G.D.A.; Stegeman, J.A.; Smidt, H. Host and environmental factors affecting the intestinal microbiota in chickens. Front. Microbiol. 2018, 9, 235. [Google Scholar] [CrossRef] [Green Version]
  91. Rinninella, E.; Raoul, P.; Cintoni, M.; Franceschi, F.; Miggiano, G.A.D.; Gasbarrini, A.; Mele, M.C. What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases. Microorganisms 2019, 7, 14. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Doughari, H.J.; Ndakidemi, P.A.; Human, I.S.; Benade, S. The ecology, biology and pathogenesis of Acinetobacter spp.: An overview. Microbes Environ. 2011, 26, 101–112. [Google Scholar] [CrossRef] [Green Version]
  93. Garrity, G. Bergey’s Manual of Systematic Bacteriology, 2nd ed.; Garrity, G., Ed.; Springer: New York, NY, USA, 2005; Volume 2: The Proteobacteria. Part B: Gammaproteobacteria, 2816p. [Google Scholar]
  94. Farag, I.F.; Davis, J.P.; Youssef, N.H.; Elshahed, M.S. Global patterns of abundance, diversity and community structure of the Aminicenantes (candidate phylum OP8). PLoS ONE 2014, 9, e92139. [Google Scholar] [CrossRef] [PubMed]
  95. Krieg, N.R.; Staley, J.T.; Brown, D.R.; Hedlund, B.P.; Paster, B.J.; Ward, N.L.; Ludwig, W.; Whitman, W.B. Bergey’s Manual of Systematic Bacteriology; Springer: New York, NY, USA, 2010; Volume 4: The Bacteroidetes, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae, and Planctomycetes, 949p. [Google Scholar]
  96. Tamaki, H. Cultivation Renaissance in the post-metagenomics era: Combining the new and old. Microbes Environ. 2019, 34, 117–120. [Google Scholar] [CrossRef] [Green Version]
Animals 11 00448 g001 550
Figure 1. Differences in the mean number of eggs (A) and mean egg weight (B) produced by control and experimental laying hens. Control hens were fed a basal diet while experimental ones were fed a basal diet supplemented with a commercial Alliaceae extract. In both cases, the number of eggs and egg weight were significantly higher in the experimental group fed the Alliaceae extract. Whiskers show 95% confidence interval and asterisks indicate outliers. Outliers are represented with *.
Figure 1. Differences in the mean number of eggs (A) and mean egg weight (B) produced by control and experimental laying hens. Control hens were fed a basal diet while experimental ones were fed a basal diet supplemented with a commercial Alliaceae extract. In both cases, the number of eggs and egg weight were significantly higher in the experimental group fed the Alliaceae extract. Whiskers show 95% confidence interval and asterisks indicate outliers. Outliers are represented with *.
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Animals 11 00448 g002 550
Figure 2. Changes in the number of eggs (A) and egg weight (B) produced by control laying hens (in blue) and laying hens experimentally supplemented with a commercial Alliaceae extract (in red) during 30 days of the experimental period. Regression lines and 95% CI are also shown.
Figure 2. Changes in the number of eggs (A) and egg weight (B) produced by control laying hens (in blue) and laying hens experimentally supplemented with a commercial Alliaceae extract (in red) during 30 days of the experimental period. Regression lines and 95% CI are also shown.
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Animals 11 00448 g003 550
Figure 3. Bar plot of the relative bacterial abundance at the phylum level in different gut regions of laying hens and treatments. Control refers to laying hens fed a basal diet while Garlicon refers to experimental laying hens fed a basal diet supplemented with a commercial Alliaceae extract.
Figure 3. Bar plot of the relative bacterial abundance at the phylum level in different gut regions of laying hens and treatments. Control refers to laying hens fed a basal diet while Garlicon refers to experimental laying hens fed a basal diet supplemented with a commercial Alliaceae extract.
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Animals 11 00448 g004 550
Figure 4. Linear Discriminant Analysis Effect Size (LEfSe) analyses showing bacterial genera (outer circles in the trees) and phyla (inner circles in the tree) that differed significantly between control hens and those supplemented with a commercial Alliaceae extract, in the ileum and cecum. Green bars and dots indicate a significant increase in relative abundance in the supplemented groups while red bars and dots showed a significant decrease.
Figure 4. Linear Discriminant Analysis Effect Size (LEfSe) analyses showing bacterial genera (outer circles in the trees) and phyla (inner circles in the tree) that differed significantly between control hens and those supplemented with a commercial Alliaceae extract, in the ileum and cecum. Green bars and dots indicate a significant increase in relative abundance in the supplemented groups while red bars and dots showed a significant decrease.
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Figure 5. Principal Coordinate Analysis based in Unweighted UniFrac (A) and Weighted UniFrac (B) distance matrixes exploring the effects in the bacterial gut community of the supplementation with a commercial Alliaceae extract in laying hens’ diet (red: ileum, treated hens; orange: cecum, treated; blue: ileum, control; green: cecum, control). Circles surround samples from similar gut region and treatment. Percentages show the proportion of variance explained by each axis.
Figure 5. Principal Coordinate Analysis based in Unweighted UniFrac (A) and Weighted UniFrac (B) distance matrixes exploring the effects in the bacterial gut community of the supplementation with a commercial Alliaceae extract in laying hens’ diet (red: ileum, treated hens; orange: cecum, treated; blue: ileum, control; green: cecum, control). Circles surround samples from similar gut region and treatment. Percentages show the proportion of variance explained by each axis.
Animals 11 00448 g005
Table
Table 1. General Linear Models exploring the effects of treatment and date in egg productivity (Number of eggs) and size (mean egg size) of laying hens. Treated hens received a basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05, which are in bold. Degrees of freedom (d.f.) are also shown. * means interaction term.
Table 1. General Linear Models exploring the effects of treatment and date in egg productivity (Number of eggs) and size (mean egg size) of laying hens. Treated hens received a basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05, which are in bold. Degrees of freedom (d.f.) are also shown. * means interaction term.
ModelVariablesd.f.Fp
Egg number1Treatment1,2720.10<0.001
Date1,271.070.310
Treatment1,26<0.010.991
2Date1,261.460.238
Treatment * Date1,2610.820.003
Mean egg weight3Treatment1,2716.93<0.001
Date1,272.890.089
Treatment1,260.040.840
4Date1,262.440.119
Treatment * Date1,265.560.019
Table
Table 2. General Linear Models exploring the effects of 30-days treatment in alpha diversity indexes in ileum and cecum of laying hens. Treated hens received in their basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05. Degrees of freedom (d.f.) are also shown.
Table 2. General Linear Models exploring the effects of 30-days treatment in alpha diversity indexes in ileum and cecum of laying hens. Treated hens received in their basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05. Degrees of freedom (d.f.) are also shown.
Alpha Diversity IndexControlExperimentald.f.Fp
Ileumsub-OUT richness165.6 (13,12)162.25 (10.37)1,110.040.845
Faith’s diversity index20.92 (1.51)20.56 (1.19)1,110.030.857
Evenness0.79 (0.03)0.74 (0.02)1,112.200.167
Shannon’s diversity index5.79 (0.26)5.40 (0.21)1,111.410.260
Cecumsub-OUT richness173.20 (13.66)172.12 (10.80)1,11<0.010.952
Faith’s diversity index21.34 (1.09)20.96 (0.86)1,110.080.789
Evenness0.67 (0.04)0.69 (0.03)1,110.180.682
Shannon’s diversity index4.97 (0.33)5.11 (0.26)1,110.120.737
Table
Table 3. General Linear Models exploring the effects of 30-days treatment in beta diversity indexes in ileum and cecum of laying hens. Treated hens received a basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05, which are in bold. Degrees of freedom (d.f.) are also shown. * means interaction term.
Table 3. General Linear Models exploring the effects of 30-days treatment in beta diversity indexes in ileum and cecum of laying hens. Treated hens received a basal diet supplemented with a commercial Alliaceae extract. Significant values were set at 0.05, which are in bold. Degrees of freedom (d.f.) are also shown. * means interaction term.
Beta Diversity IndexFactorsd.f.Fp
GutUnweighted UniFfracTreatment1,223.53<0.001
Gut1,221.960.002
Gut * Treatment1,220.760.898
Weighted UniFfracTreatment1,224.270.003
Gut1,226.55<0.001
Gut * Treatment1,221.030.391
IleumUnweighted UniFfracTreatment1,111.81<0.001
Weighted UniFfracTreatment1,112.210.056
CecumUnweighted UniFfracTreatment1,112.630.002
Weighted UnifracTreatment1,113.110.020
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Rabelo-Ruiz, M.; Ariza-Romero, J.J.; Zurita-González, M.J.; Martín-Platero, A.M.; Baños, A.; Maqueda, M.; Valdivia, E.; Martínez-Bueno, M.; Peralta-Sánchez, J.M. Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum. Animals 2021, 11, 448. https://doi.org/10.3390/ani11020448

AMA Style

Rabelo-Ruiz M, Ariza-Romero JJ, Zurita-González MJ, Martín-Platero AM, Baños A, Maqueda M, Valdivia E, Martínez-Bueno M, Peralta-Sánchez JM. Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum. Animals. 2021; 11(2):448. https://doi.org/10.3390/ani11020448

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Rabelo-Ruiz, Miguel, Juan José Ariza-Romero, María Jesús Zurita-González, Antonio Manuel Martín-Platero, Alberto Baños, Mercedes Maqueda, Eva Valdivia, Manuel Martínez-Bueno, and Juan Manuel Peralta-Sánchez. 2021. "Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum" Animals 11, no. 2: 448. https://doi.org/10.3390/ani11020448

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