Effect of an Herbal Mixture of Oregano, Garlic, Sage and Rock Samphire Extracts in Combination with Tributyrin on Growth Performance, Intestinal Microbiota and Morphology, and Meat Quality in Broilers

Abstract

The present study investigated the effects of two feed additives, the first containing an herbal mixture of oregano, garlic, sage, and rock samphire extracts and the second containing tributyrin (glyceryl tributyrate) when fed to broiler chickens. A total of 360 one-day-old chicks were randomly allocated to four treatments (6 replicate pens of 15 chicks). One treatment served as the unsupplemented control, whereas the feeds of the other three treatments were supplemented either with the herbal additive (3 g/kg), the tributyrin additive (1 g/kg), or both additives. The duration of the trial was 37 days. Data were collected on growth performance, intestinal microbiota and morphology, and some meat quality parameters. The combined supplementation improved (p < 0.05) weigh gain, feed conversion ratio, and the European Efficiency Factor. In the cecum, the combined supplementation lowered (p < 0.05) the microbial populations of aerobes, anaerobes, Escherichia coli, total Enterobacteriaceae, and Clostridium spp. compared to the other treatments. Fecal coccidial oocyst counts were also reduced (p < 0.01) by the combined supplementation. The herbal mixture supplementation improved (p < 0.05) breast and thigh meat resistance to oxidation. In conclusion, the combined dietary supplementation with the examined feed additives could be utilized to improve the performance and intestinal health of broiler chickens.

1. Introduction

Chickens are an essential source of animal protein and meat products that play a vital role in human nutrition and development, especially in high gross domestic product (GDP) countries [1,2]. It is widely acknowledged that the human population is growing fast, particularly in countries where food consumption is expected to be inadequate and disproportional to that growth, and by 2050 it is anticipated to increase to 9.5 billion worldwide [2]. Thus, humanity will have to ensure up to 60% additional food resources compared to the current levels of 8.5 billion tons per year to feed such a population [3]. At the same time, grassland degradation, biodiversity, and greenhouse gas production due to ruminants are particular concerns that have to be addressed to achieve sustainable food production [3]. The broiler meat industry has been expanding rapidly in the last decades, in response to the constant increase of global meat consumption. An estimate growth of 121% in poultry meat production is expected, far more significant to 66% and 43% of beef and pork meat, respectively [1]. This high demand in poultry meat products is also due to their wide acceptance as healthier alternatives characterized by higher protein and lower lipid content compared to other meats and to their cost-effective productivity values in most cultures, religions, and traditions [4].

To achieve these demands of the broiler industry, many feed additives have been employed to improve productivity and other traits, along with improvements in genetics and management [5]. Extensive research has acknowledged the importance of phytobiotics and other feed additives such as organic acids in animal nutrition and sustainability. Currently, the use of dietary growth promoting antibiotics has been banned or placed under major restrictions in most developed countries worldwide, due to their contribution to the development of microbial resistance in humans and animals [6,7]. In chickens a typical example is the presence of the extended-spectrum beta-lactamase (ESBL) bacteria, a major antibiotic–resistance concern for public health [8]. Moreover, consumers are aware of the importance of quality food and prefer natural products, free of any pharmaceutical residues in the food chain. Phytobiotics, as plant derived material, their extracts, and/or their essentials oils, have played an important part in animal nutrition, with numerous studies focusing on their positive effect in feed palatability and intake, anti-inflammatory, antioxidant and/or antimicrobial activity, intestinal function, and growth performance [9,10]. Essential oils derived from oregano (Origanum spp.) and sage (Salvia triloba) plants, grown abundantly in the Mediterranean area, contain potent antioxidant substances that have the ability to reduce the phenomenon of oxidative stress in tissues and/or serum samples, while improving the physical characteristics of chicken meat [11]. These effects are attributed to their rich content in phenolic compounds [12]. Moreover, the active compounds of garlic (Allium sativa) and its derivatives, such as flavonoids and organosulphurs, have shown to possess antioxidant capacity, as they were found to deteriorate lipid oxidation in chickens, along with various therapeutic applications [13,14]. Another plant of interest, rock samphire or sea fennel (Crithmum maritimum), is grown in coastal dunes of the Mediterranean basin [15]. Recently, components of its hydro-ethanolic extracts were found to possess antioxidant and antimicrobial properties [16].

Another category of important non-antibiotic feed additives in broiler nutrition are the organic acids (OAs) which represent promising feeding strategy in the industry [17]. The inclusion of OAs in animal diet has been involved in several studies evaluating intestinal tract parameters, immunity, and production performance [18,19]. Some of the most important OAs involved in animal nutrition are butyric, citric, propionic, ascorbic, formic, acetic, benzoic, and fumaric acids [20,21]. Organic acids in their salt or ester forms have a wide application in animal nutrition, as they have the ability to positively alter the immune responses, possess antioxidant and anti-inflammatory capacity, thus maintaining the balance of gastrointestinal homeostasis and the epithelial integrity, and are involved in the energy metabolism of monogastric animals [20,22]. Their mode of action is at least partially linked to the reduction of intestinal pH, which supports the growth of advantageous strains such as lactobacilli while decreasing pathogen populations, such as Escherichia coli and/or Salmonella, therefore affecting the intestine microbiota [20,23]. Since the health status of farm animals is of high importance and directly correlated with animal welfare traits, performance, and farm profitability, the intestinal inflammation and dysfunction, along with altered gut microbiota, could represent major issues in the poultry industry [22]. Furthermore, few reports have shown a synergistic effect of encapsulated butyric acid, when supplied in combination with other compounds such as oregano and/or attapulgite clay, on broiler performance and intestinal health [24,25].

The aim of the present study was to investigate the potential benefits of the combined use of an herbal feed additive (containing oregano, garlic, sage, and rock samphire) with another feed containing an organic acid (glyceryl tributyrate) on growth performance, intestinal microbiota and morphology, and some meat quality parameters of broiler chickens.

2. Materials and Methods

2.1. Animals, Diets and Experimental Design

The protocol of the experimental project “Innochicken” was co-financed by the European Regional Development Fund (ERDF) under the Operational Program “Epirus 2014–2020”, NSRF 2014–2020. Project Code: HΠ1AB-0028192. During the experimental trial, the broiler chickens were managed in compliance with local ethical practices and procedures [26] and following the recommendations for broiler welfare [27].

Three hundred sixty one-day-old male Ross-308 chicks (initial body weight 45.3 ± 0.7 g) were obtained from the PINDOS APSI hatchery and reared in a commercial poultry farm in Gavria, Arta, (latitude 38.617°, longitude 20.767°), Epirus, Greece, during the period of October–December 2019. There were 4 treatment groups, each with 6 replicate pens (length 1 m; width 1.1 m) of 15 chicks. The stocking density was calculated to be 15 birds per m² (area of 1.1 m² per pen). Throughout the experimental period, commercial breeding and management procedures were applied. Both natural and artificial lighting were used to achieve light of 23 h for the first two days, 16 h from day 3 to day 14, and 21 h from day 15 to slaughter. Ambient temperature and humidity were carefully controlled by a computer monitoring system. All chicks were vaccinated against Newcastle disease, infectious bronchitis, and infectious bursal disease (Gumboro) at the hatchery. Ad libitum feeds and drinking water were given to all broilers throughout the trial.

Control treatment (CONTR) chickens were fed commercial typical corn and soybean meal based rations in mash form (

Table 1. Ingredients and nutrient content of the diets fed to the control treatment.

Ingredients (%)	Starter Days 1–14	Grower-Finisher Days 15–37
Maize	49.108	51.339
Wheat	10.000	10.000
Soybean meal (47% crude protein)	33.547	30.715
Soybean oil	2.623	3.769
Salt	0.325	0.315
Sodium carbonate	0.074	0.077
Limestone (Calcium carbonate)	1.512	1.390
Dicalcium phosphate	1.516	1.300
Lysine HCl	0.422	0.327
Methionine DL	0.434	0.374
Threonine	0.189	0.144
Vitamin and mineral premix 1	0.250	0.250
Total	100.000	100.000
Nutrient content
Metabolisable energy (Kcal/kg)	3090.0	3180.0
Crude protein (%)	23.50	22.50
Crude fat (%)	5.50	5.80
Total lysine (%)	1.50	1.40
Total methionine (%)	1.40	1.20
Total calcium (%)	1.00	1.00
Available phosphorus (%)	0.60	0.60

¹ Supplying per kg feed: 13,000 IU vitamin A, 4000 IU vitamin D3, 40 mg vitamin E, 9 mg vitamin K, 3 mg thiamine, 7 mg riboflavin, 6 mg pyridoxine, 0.035 mg vitamin B12, 40 mg niacin, 13 mg pantothenic acid, 1.5 mg folic acid, 0.13 mg biotin, 340 mg choline chloride, 55 mg Zn, 155 mg Mn, 20 mg Fe, 12 mg Cu, 0.2 mg Co, 1 mg I, 0.2 mg Se, and phytase 500 FTU.

), which did not contain anticoccidials or antibiotics, formulated based on breeder recommendations [28,29]. The diets of the other three treatments were further supplemented with either the tested herbal mixture (3 g/kg; HERB), the tested butyrate additive (1 g/kg; BUTYR), or both the herbal additive (3 g/kg) and the butyrate additive (1 g/kg) together (HERB&BUTYR). The herbal mixture was formulated to provide in the feed: 50 ppm oregano essential oil; 5 ppm garlic essential oil; 1 g/kg dried sage; and 1 g/kg dried rock samphire. The herbal material was supplied by a local farm in Palaiohori, Filiates, Thesprotia, Greece. The butyrate additive contained 100% glyceryl tributyrate (“Butiphorce 1065”, NuSana, Land van Cuijk, The Netherlands).

The broiler weights were determined on days 1, 12, 24, and 37. Data on feed consumption and mortality were collected daily. The “European Production Efficiency Factor (EPEF)” was calculated for the overall trial [30], using the formula:

EPEF = [Average daily weight gain (g) × Survival rate (%)]/[Feed conversion ratio × 10]

On the last day of the trial (day 37), all broilers were humanely slaughtered under commercial conditions at a nearby abattoir. Four chickens were randomly chosen and processed from each replicate pen (24 per treatment; 96 total). During processing, the gastrointestinal tracts of the selected chickens were carefully removed for further analysis.

2.2. Gastrointestinal Tract Sampling

To collect the gastrointestinal tissues, initially the abdomen of each bird was cleaned with 70% (v/v) ethanol and skin incisions were aseptically performed to access the intestine. Then, the jejunum and the caeca of each chicken were separated and dissected using a sterile scalpel, which was also used to gently scrape off the mucus layer from the intestinal content of each incision site and transfer it to a sterile container and stored.

2.3. Microbiological Analysis

For the bacterial isolation, enumeration, and identification, 1 g of intestinal content from each bird was weighted and homogenized with 9 mL of sterile peptone water solution 0.1%. Miles and Misra plate method (surface drop) was used for bacterial enumeration in which each sample was serially diluted via 10-fold dilutions (from 10⁻¹ to 10⁻¹²) using standard 96-well microplates, while dilutions were plated onto selective mediums which were incubated properly [31]. Total aerobic and anaerobic bacterial counts were determined using plate count agar medium (PCA) (Oxoid, Basingstoke, UK), while plates were incubated at 30 °C aerobically for 48 h and at 37 °C anaerobically for 48–72 h, respectively. For the isolation and enumeration of E. coli and total Enterobacteriaceae, MacConkey agar (Merck, Darmstadt, Germany) and Violet Red Bile Glycose (VRBG) agar (Merck) were used, and plates were both incubated aerobically at 37 °C for 24–48 h. De Man, Rogosa, and Sharpe (MRS) agar (Oxoid) was used for the isolation and enumeration of Lactobacillus species, while plates were incubated at 37 °C for 48–72 h under anaerobic condition. For the isolation and enumeration of Clostridium spp., Tryptose Sulfite Cycloserine (TSC) agar was used (Merck) and plates were incubated at 37 °C for 24–48 h in anaerobic conditions. After incubation time, typical colonies from an appropriate dilution were counted and expressed as log colony-forming units per 1 g wet weight sample (CFU/g). In addition, typical colonies were characterized morphologically by microscopy, gram staining, catalase, and oxidase tests and sub-cultured in order to obtain pure cultures. Lactobacilli were identified using API 50 CHL kits according to the manufacturer’s instructions and API LAB Plus software version 3.3.2 (Bio-Merieux, Marcy-l’Étoile, France). Identification of Clostridium spp. were performed by VITEK 2 ANC card, while identification of E. coli by GN identification card was performed by API LAB Plus software version 3.3.2 (bioMérieux, Marcy l’Etoile, France). ANC and GN identification cards were used in conjunction with VITEK 2 system (bioMérieux, Marcy l’Etoile, France) [32].

2.4. Coccidial Oocysts Count

On the 28th and 37th day of the trial, fresh faecal samples were taken from chickens from all experimental pens to determine coccidial oocyst output. The McMaster technique was applied to identify and count coccidian oocysts in these faecal samples [33,34]. The identification of unsporulated oocysts of Eimeria spp. were based on their morphological features (oocyst shape, mean oocyst length, and mean oocyst width after microscopic examination).

2.5. Intestinal Morphometric Analysis

To perform the morphometric analysis of the samples from the small intestine, the methodology described by Gava et al. [35] was initially followed. Then, photographic images were taken under light microscopy, using a Nikon microscope coupled with a Microcomp integrated digital imaging analysis system (Nikon Eclipse 200, Tokyo, Japan). Images were analysed with the “Image-Pro Plus” analysis software. Villus height (VH) and crypt depth (CD) were measured as the mean of 10 collected values per sample (Figure 1).

2.6. Meat Chemical Analysis

The carcasses of the selected birds were initially processed according to the slaughterhouse commercial procedures. Then, breast and thigh meat samples were collected from each carcass; they were carefully skinned, deboned, and grounded using an industrial large meat grinder. Then, sub-samples of 200 g were analyzed for moisture, crude protein, fat, and ash content by near infra-red spectroscopy, using a FoodScanTM Lab (FOSS, Hillerød, Denmark) as described in reference method AOAC 2007.04 [36,37].

2.7. Meat lipid Oxidation Analysis

Breast and thigh meat samples were processed according to Ahn et al. [38] with minor modifications to determine their lipid oxidation status during refrigerated storage, with a UV spectrophotometer (UV 1700 PharmaSpec, Shimadzu, Kyoto, Japan) set at 532 nm. The lipid oxidation of the samples was evaluated as “2-thiobarbituric acid-reactive substances (TBARS)” values, expressed as ng of malondialdehyde (MDA) per g of sample.

2.8. Statistical Analysis

The experimental study design was RCBD (random complete block design). In all measurements, the replication (pen) was considered as the experimental unit. Experimental data were evaluated using the one-way ANOVA (general linear model) and the Kruskal–Wallis tests of the SPSS statistical package (version 20.0) [39]. Microbiology data were log-transformed (Log10) prior to analysis and data homogeneity was examined with the Levene’s test. The threshold for significance was set at 5% (p ≤ 0.05).

3. Results

The effects of the dietary supplementation on broiler chicken performance parameters are presented in

Table 2. Effect of combined herbal and organic acid dietary supplementation on the broiler chicken performance parameters.

Live Body Weight on Day (g)	CONTR	HERB	BUTYR	HERB&BUTYR	SEM	p
1	45.0	45.2	45.7	45.4	0.13	0.254
12	303.6	314.5	344.0	310.1	5.54	0.185
24	923.1	1016.6	982.2	960.1	13.90	0.160
37	1685.9 a	1833.9 ab	1794.3 ab	1846.9 b	19.07	0.023
Feed intake during period (g)
1–12 days	322.7	323.8	324.4	324.5	0.00	1.000
13–24 days	1007.9 b	1004.1 b	968.3 a	985.7 ab	4.15	<0.001
25–37 days	1224.6	1318.9	1295.8	1275.5	24.11	0.628
1–37 days	2574.8	2662.6	2603.9	2620.4	25.49	0.675
Feed conversion ratio during period (g feed/g weight gain)
1–12 days	1.249	1.204	1.113	1.228	0.020	0.332
13–24 days	1.630	1.439	1.529	1.535	0.027	0.133
25–37 days	1.608	1.614	1.602	1.464	0.036	0.423
1–37 days	1.569 b	1.489 ab	1.494 ab	1.456 a	0.013	0.043
European efficiency factor
Day 37	270.2 a	321.7 ab	315.1 ab	324.4 b	7.62	0.026

CONTR: control non-supplemented treatment; HERB: feed supplemented with herbal mixture at 3 g/kg; BUTYR: feed supplemented with glyceryl tributyrate at 1 g/kg; HERB&BUTYR: feed supplemented with herbal mixture at 3 g/kg and glyceryl tributyrate at 1 g/kg. SEM: standard error of the means. ^a,b: values in the same row without superscripts in common differ significantly (p ≤ 0.05).

. The combined supplementation of the herbal and the organic acid additives (HERB&BUTYR) significantly increased (p < 0.05) the final body weight, compared to the unsupplemented control (CONTR), at the end of the trial (day 37). The feed intake was found to be significantly lower (p < 0.001) for the BUTYR treatment compared to the CONTR and HERB treatments during the period of 13–24 days; however, this effect was not noted for the other periods or the overall trial. In addition, overall feed conversion ratio (days 1–37) was significantly lower (p < 0.05) and the European Efficiency Factor was significantly improved (p < 0.05) for the HERB&BUTYR treatment compared to the unsupplemented CONTR treatment.

Table 3. Effect of combined herbal and organic acid dietary supplementation on the broiler chicken intestinal microbiota.

Jejunum Microbiota (Log CFU/g Digesta)	CONTR	HERB	BUTYR	HERB&BUTYR	SEM	p
Total aerobic counts	6.263	6.405	6.604	6.193	0.142	0.748
Total anaerobic counts	8.143	7.851	8.361	8.209	0.116	0.479
Escherichia coli	3.530 a	5.030 ab	5.217 b	3.941 ab	0.191	0.029
Total Enterobacteriaceae	3.829 a	5.341 ab	5.419 b	4.511 ab	0.172	0.017
Lactobacillus spp.	7.576	7.562	7.873	7.694	0.154	0.883
Clostridium spp.	2.882 b	2.555 a	2.465 a	2.303 a	0.034	<0.001
Cecum microbiota (Log CFU/g digesta)
Total aerobic counts	7.751 ab	8.239 b	7.748 ab	7.029 a	0.093	0.002
Total anaerobic counts	8.824 b	8.216 a	8.692 ab	8.242 a	0.063	0.010
Escherichia coli	7.440 b	7.172 b	7.427 b	6.176 a	0.105	0.001
Total Enterobacteriaceae	7.564 b	7.289 b	7.660 b	6.531 a	0.096	0.002
Lactobacillus spp.	8.251	8.271	7.918	8.201	0.119	0.706
Clostridium spp.	4.869 b	4.816 b	5.371 b	3.765 a	0.122	0.001
Oocyst counts in feces (Log/g)
Day 28	3.989 c	3.672 b	3.835 bc	3.304 a	0.037	<0.001
Day 37	4.007 b	3.475 a	3.645 ab	3.388 a	0.053	0.003

CONTR: control non-supplemented treatment; HERB: feed supplemented with herbal mixture at 3 g/kg; BUTYR: feed supplemented with glyceryl tributyrate at 1 g/kg; HERB&BUTYR: feed supplemented with herbal mixture at 3 g/kg and glyceryl tributyrate at 1 g/kg. SEM: standard error of the means. ^a,b: values in the same row without superscripts in common differ significantly (p ≤ 0.05).

shows the effects of the dietary supplementation on the intestinal microbiota. In the jejunum, E. coli and total Enterobacteriaceae were significantly increased (p ≤ 0.05) in the BUTYR treatment compared to the CONTR; Clostridium spp. was significantly decreased (p ≤ 0.001) in the three supplemented treatments compared to the CONTR; and total aerobes and anaerobes were not significantly (p > 0.05) affected. In the cecum, total aerobes were significantly decreased (p ≤ 0.01) in the HERB&BUTYR treatment compared to the HERB treatment; total anaerobes were significantly decreased (p ≤ 0.01) in the HERB and HERB&BUTYR treatments compared to the CONTR; and E. coli, total Enterobacteriaceae, and Clostridium spp. were significantly decreased (p ≤ 0.001; p ≤ 0.01; and p ≤ 0.001, respectively) in the HERB&BUTYR treatment compared to the other three treatments. In addition, oocysts analysis from the broiler feces on day 28 showed that the HERB&BUTYR treatment had significantly lower (p < 0.001) counts compared to all other treatments, while also HERB treatment had lower counts compared to the CONTR. In addition, the oocyst analysis on day 37 showed that the HERB treatments and HERB&BUTYR treatments had significantly lower (p < 0.01) counts compared to the CONTR.

The results of the broiler chicken jejunum morphology analysis are given in

Table 4. Effect of combined herbal and organic acid dietary supplementation on the broiler chicken jejunum morphology.

Jejunum Morphology	CONTR	HERB	BUTYR	HERB&BUTYR	SEM	p
Jejunum villus height (μm)	764.6	754.1	710.3	697.5	14.02	0.269
Jejunum crypt depth (μm)	196.4	197.4	203.1	183.0	8.31	0.889
Jejunum Villus height/Crypt depth	3.976	3.931	3.535	3.819	0.1578	0.810

CONTR: control non-supplemented treatment; HERB: feed supplemented with herbal mixture at 3 g/kg; BUTYR: feed supplemented with glyceryl tributyrate at 1 g/kg; HERB&BUTYR: feed supplemented with herbal mixture at 3 g/kg and glyceryl tributyrate at 1 g/kg. SEM: standard error of the means.

. No significant effects (p > 0.05) were identified in jejunum villus height, jejunum crypt depth, or the villus height to crypt depth ratio.

The effects of the dietary supplementation on the chemical composition and oxidative stability of the meat are presented in

Table 5. Effect of combined herbal and organic acid dietary supplementation on the broiler chicken meat chemical composition and oxidative stability.

Breast Meat Chemical Composition	CONTR	HERB	BUTYR	HERB&BUTYR	SEM	p
Moisture %	74.96	74.78	74.36	75.01	0.122	0.271
Crude protein %	23.84	22.34	22.55	22.24	0.300	0.257
Crude fat %	1.57 a	1.94 ab	2.03 ab	2.40 b	0.082	0.028
Ash %	0.76	0.76	0.74	0.74	0.024	0.527
Breast meat lipid oxidation
Day 1 of storage (ng MDA/g)	28.8 ab	20.4 a	44.6 b	21.9 ab	1.668	0.020
Thigh meat chemical composition
Moisture %	73.65	72.44	72.09	71.94	0.321	0.303
Crude protein %	19.87	18.78	19.66	18.50	0.247	0.207
Crude fat %	6.24	8.08	7.77	8.70	0.389	0.240
Ash %	0.64	0.71	0.60	0.70	0.022	0.250
Thigh meat lipid oxidation
Day 1 of storage (ng MDA/g)	21.8 b	14.2 a	28.5 c	24.3 b	0.685	<0.001

CONTR: control non-supplemented treatment; HERB: feed supplemented with herbal mixture at 3 g/kg; BUTYR: feed supplemented with glyceryl tributyrate at 1 g/kg; HERB&BUTYR: feed supplemented with herbal mixture at 3 g/kg and glyceryl tributyrate at 1 g/kg. SEM: standard error of the means. ^a,b: values in the same row without superscripts in common differ significantly (p ≤ 0.05).

. Regarding the breast meat, a significantly (p ≤ 0.05) higher amount of fat was found in the HERB&BUTYR treatment, compared to the unsupplemented CONTR. Moreover, lipid oxidation (expressed as ng MDA/g meat) was found to be significantly lower (p ≤ 0.05) in the breast meat of the HERB treatment compared to the BUTYR treatment. Regarding the thigh meat, no significant effects (p > 0.05) were identified in the chemical composition. However, the lipid oxidation of the thigh meat was significantly lower (p ≤ 0.001) in the HERB treatment compared to all other treatments, as well as lower in the CONTR and HERB&BUTYR treatments, compared to the BUTYR treatment.

4. Discussion

Feed additives have been widely employed in broiler nutrition to increase performance characteristics, improve farm animal welfare, and ultimately achieve sustainability in addition to high productivity [20,40]. Herbal plant and organic acid-based feed additives have been evaluated and considered among the most promising supplements for poultry and thus have been screened in numerous studies on broiler performance, sometimes with conflicting results [41,42].

The European Production Index related to the outcome of both performance and health. One way to improve overall productivity is the dietary use of functional feed additives that support weight gain by supporting the digestive and immune systems. Additives with beneficial properties include aromatic plants and their extracts that have long been used as alternatives to antibiotic growth promoters, mainly due to their valuable constituents such as polyphenols [43]. The oregano essential oil used in our study was found to be rich in carvacrol (75.06%) and thymol (6.56%), with both constituents playing a significant role on its antioxidant capacity [44]. Oregano as a feed supplement has been found to possess antimicrobial applications against poultry and human pathogens [45]. The principal phytochemicals of garlic that display antioxidant and antibacterial activity are oil-soluble organosulfur compounds such as allicin and/or allyl sulfides [46,47]. The most prominent constituents of garlic essential oil were diallyl sulfides (diallyl disulfide and trisulfide). In addition, garlic is known for its effects on the immune response and the regulation of the gastrointestinal microbiome. Garlic polysaccharides have been found to have the capacity to reduce the expression of inflammatory factors and improve the colon tissue integrity and microbiota [48]. Sage plants are also rich in polyphenols and flavonoids including catechin, rutin, caffeic, and rosmarinic acids, while their volatile oils contain mainly monoterpenes and sesquiterpenes such as carnosic acid [49]. However, the concentration of those constituents may differ to a great extent depending on the Salvia species. Aqueous sage extract has shown significant inhibitory activity against different bacterial species such as Bacillus mycoides, Bacillus subtilis, Enterobacter cloacae, and Proteus spp. [50], whereas the hydroalcoholic extract has demonstrated strong inhibitory effect on Streptococcus mutans, Lactobacillus rhamnosus, and Actinomyces viscosus [51]. Thus, sage extract has been suggested as a valid alternative source to traditional antibiotics [52]. Rock samphire is rich in polyphenols such as chlorogenic acid and flavonoids including rutin, cirsiliol, and quercetrin [16]. Rock samphire essential oil components have been shown to inhibit the growth of food-borne bacteria, including E. coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Listeria innocua, and others [53].

Tributyrin is a triglyceride ester of butyric acid and glycerol [54]. It is naturally found in butter and margarine. Butyric acid is also naturally produced by fermenting bacteria in the caecum [55]. It has been suggested that luminal butyric acid is linked to the endocrine regulation of the digestive tract affecting enteroendocrine cells and overall nutrient digestibility [56]. When used as feed additive, the activity of butyric acid in its free form is limited to the crop, proventriculus, and gizzard of the broilers. However, protected forms of butyric acid such as its esters are hydrolyzed by the pancreatic lipase to glycerol tributyrate and dibutyrate that are effective in the small intestine [54,57]. Dietary butyric acid and its esters have been reported to limit the growth of pathogens [58], stimulate feed intake, and improve digestion by stimulating pancreatic secretion [59].

The potential benefits of dietary herbal extracts and organic acids on broiler performance have been widely examined. Nevertheless, published data on the combined supplementation of these two additives are still quite scarce. Studies evaluating combinations of plant essential oils with organic acids in poultry nutrition showed that these combinations increased growth rates and/or feed utilization [19,24,60,61,62,63], which is in agreement with the results of our study. It should be noted that such positive effects are not always reported [64,65,66]. This inconsistency can be explained in part by the wide variety of the tested herbal or organic acid material, as well as the different inclusion rates. In addition, the active substances of plant essential oils and dissociated organic acids are quickly absorbed in the crop and stomach of the birds, thus exerting their direct effects in the anterior part of the gastrointestinal system [67]. Additionally, some phytobiotics increase feed palatability and stimulate the production of digestive enzymes [61,68]. Most dietary organic acids are weak acids that only partially dissociate in aquatic solution and the percentage of dissociation depends on the pH of the gastrointestinal tract [69]. For these reasons, protective technologies, such as encapsulation of the active ingredients, are under examination [17,60]. These methods lower the rate of absorption or degradation of the active ingredients in the anterior part of the gastrointestinal tract and allow them to reach and exert their effects on the latter parts of the intestine such as the ileum and the ceca of the birds, directly affecting the intestinal microbiota as well as the intestinal epithelium.

An important indicator of intestinal health is the integrity of the intestinal epithelium. The villus size, absorptive area, and thickness of the mucus layer regulate the transport of molecules from the intestinal lumen through the epithelium into the bloodstream [70]. Intestinal disease severely limits the villus length and uniformity because bacterial toxins rapidly damage the membranes of the epithelial cells [71,72]. The replacement of the intestinal epithelium poses an important energy cost for the animal. Decreased crypt depth is linked to lower tissue turnover and reduced secretion [71,72]. It has been reported that dietary plant essential oils can increase villus height and cell proliferation in chickens [24,73,74]. Moreover, dietary organic acids can improve villus height in the small intestine, directly by causing enterocyte hyperplasia or limiting apoptosis [63,75] or indirectly through the modification of the microbiota balance [19,76]. In our study, no significant differences were noted on villus height and crypt depth between the supplemented treatment and the unsupplemented control, which could reflect the overall high health status of both the supplemented and unsupplemented treatment groups.

In the last two decades, a large effort has been made to examine natural feed ingredients as potential alternatives to antimicrobial and anticoccidial drugs in meat type poultry [7,77,78]. Recent information on necrotic enteritis and coccidiosis in European countries elucidate their regular occurrence accompanied by major economic losses, especially after the ban of antibiotic growth promoters [79]. Although these drugs are highly effective in preventing or treating clinical disease in broilers, their overuse has caused a global alarm due to the adverse effects of their residues on the environment [12,80]. Many antimicrobials are poorly absorbed or metabolized in the gastrointestinal tract of the birds, resulting in a large amount being excreted in the feces. Subsequently, animal waste is used as fertilizer or reaches surface water, unbalancing the microbiota of natural ecosystems and increasing antibiotic resistance in animal and human pathogens [7,81]. In our study, the chicken control diet did not contain any antimicrobial or anticoccidial drugs. The combined dietary supplementation with the herbal mixture and the organic acid influenced mainly the cecum microbiota, lowering some microbial population such as E. coli, Enterobacteriaceae, and Clostridium spp. Fecal oocyst counts were also lowered in broilers receiving the combined supplementation. It should be noted that the tested broilers in our trial were not exposed to experimental Eimeria infection, but natural exposure was expected as they were reared in a commercial setting. Coccidiosis is caused by Eimeria species parasites and today it constitutes one of the biggest challenges for the broiler industry due to the ubiquity of these parasites and the increasing occurrence of anticoccidial resistance [82]. Nowadays, restrictions on the use of anticoccidial drugs increase worldwide and anticoccidial vaccination is still not widely available and affordable [83]. Extracts from medicinal aromatic plants are potential alternatives in the effort to control coccidiosis [84,85,86].

In the present study, the effect of the dietary supplements on the breast and thigh meat chemical composition was additionally evaluated. The breast meat analysis showed that the combined supplementation significantly increased fat percentage, whereas the same effect was not confirmed statistically in the thigh meat. This increase of fat content in the meat could be attributed to the improved gut health and, thus, better feed digestion that increased the metabolisable energy that remained available to the broilers for deposition in the muscle tissue. It is known that dietary composition can regulate lipid metabolism and carcass fat content, affecting key enzymes associated with lipid anabolism; however, the underlying factors have not been fully elucidated [87]. Furthermore, in the present study, resistance to oxidation was mainly affected by the herbal supplementation, whereas the organic acid supplementation seemed to trigger oxidation of lipids in the meat. It is well established that dietary herbal extracts can positively affect meat oxidative stability, influencing various mechanisms of action, i.e., improving the activity of antioxidant enzymes that counteract inflammatory agents such as reactive oxygen species, as well as protecting cells membranes from oxidation, atrophy, or breaks [88,89]. It has been reported that essential oils from Labiatae family plants such as oregano, thyme, rosemary, and others can protect refrigerated poultry products by lowering meat oxidation mainly due to the effect of their polyphenol compounds or a sparing effect on tocopherols [90,91,92]. In addition, a herbal mixture including thyme, rosemary, garlic, and others protected broiler meat from oxidative rancidity during storage [93].

5. Conclusions

Based on the results of this study, the combined dietary supplementation of two feed additives, the first containing a mixture of oregano, garlic, sage, and rock samphire ex-tracts and the second containing glyceryl tributyrate, can improve growth performance parameters of broiler chickens. Moreover, this supplementation can potentially modify beneficially the intestinal microbiota of the broilers and lower the overall oocyst count in the feces. In addition, the combined dietary supplementation seemed to increase breast meat fat content, whereas the herbal feed additive mainly improved the resistance to oxidation of the meat. Additional in vivo studies could potentially determine the optimum combinations of herbal and organic acid feed additives, to achieve sustainable and profitable broiler farming without the use of anticoccidial or ionophores.