Morphological Modulation of Broiler Organs in Response to an Organic Acid–Phytogen Composite in Healthy Broilers

Abstract

In this study, we investigated broiler growth traits, intestinal health and immune aggregates in response to supplementation of Origanum vulgare and Cinnamomum verum, citric acid and malic acid in drinking water at varying levels and durations on broiler. We randomly allocated 140 day-old broilers into four (4) groups, with each group having seven replicates (n = 5). A basal diet and water were provided to all the groups ad libitum in accordance with standard farming practices. Keeping group I as the control, group II was supplemented with 1 mL/3.3 L of water for 1–4 days, 1 mL/4 L from 15–18 days and 1 mL/4 L water from 26–29 days. Group III and IV were supplemented with 1 mL/4 L of water and 1 mL/6 L of water for 35 days, respectively. After the completion of the trial (day 35), two broilers per replicate (14 birds per group) were slaughtered to sample the visceral organs. The results demonstrated that the feed conversion ratio (FCR) and body weight gain improved (p ≤ 0.05) during the fourth and fifth week of the trial in group III in comparison to the control group. Although the small intestine villus height and villus-height-to-crypt-depth ratio improved (p ≤ 0.05) in group III, the muscularis externa thickness of the ileum was highest (p ≤ 0.05) in the control group. Similarly, the lymphatic follicular and nodular area of the immune organs improved (p ≤ 0.05) in group III. In all segments of the small intestine (duodenal, jejunal and ileal), the number of acidic and total goblet cells was highest (p ≤ 0.05) in group II. In conclusion, dietary Origanum vulgare, Cinnamomum verum, citric acid and malic acid at the dose of 1 mL/4 L of drinking water (group III) was the most effective in improving the growth performance, gut development and associated immune components in broilers.

1. Introduction

Poultry meat is an economical source of minerals, vitamins and protein for humans worldwide with unsaturated fatty acids and a low content of saturated fatty acids [1]. Poultry farming not only contributes to the gross domestic product (GDP) but is also a significant source for low-income families. The increase in demand due to higher population growth has led the poultry industry to use in-feed antibiotics (IFAs) as growth promoters at sub-therapeutic levels [2]. Although in-feed antibiotics promote the growth of broiler chickens, their residue in poultry products causes the development of antibiotic resistance in consumers, which is harmful for the health [3].

Resultantly, in 2006, the European Union prohibited the use of antibiotics as growth promoters in animal feed (EC Regulation No. 1831/2003) [4]. These developments have directed the research efforts towards identification of alternatives of IFAs for use in broiler feed.

Essential oils are phytogenic compounds that are natural bioactive secondary products of plant extracts [5]. These are being used in animal feed and poultry with or without organic acids. These essential oils carry antibacterial [6], antifungal [7], antiviral [8], antiparasitic [9], antioxidant [10] and medicinal properties [11] and function as growth enhancers [12], e.g., the essential oils of oregano, thyme, garlic, sage, cinnamon, clove and cumin [13].

On the other hand, organic acids are carboxylic acid compounds, such as propionic acid, citric acid, malic acid, formic acid, etc. [14]. They are residue free and nontoxic and are used as an alternative to infeed antibiotics in birds as approved by the European Union. Organic acids improve nutrient availability through their bactericidal effect on pathogenic bacteria. This, in turn, causes the enhanced absorption and digestibility of nutrients leading to increased growth performance [15].

A synergistic relationship between organic acids and phytobiotics has been observed when a co-supplementary diet is offered to farm birds [16]. Phytogenic essential oils interact with the bacterial cell membrane and make it more permeable, which facilitates the entrance of organic acids in bacterial cell [17]. The accumulation of organic acids in pathogenic bacterial cell reduces the intracellular pH, which disrupts cellular functions and depletes energy reservoirs as the cell attempts to re-establish homeostasis [18]. This dysfunction leads to cell death, thus, decreasing pathogenic bacteria and improving the digestive efficacy.

The O. vulgare has active ingredients, such as thymol and carvacrol, which act as a strong antioxidant and antibacterial [19] whereas C. verum has antioxidative properties. In addition, it is also widely used as a flavoring agent in commercial feed formulations for poultry and domestic animals [20]. On the other hand, citric acid and malic acid being short chain fatty acid decrease the gut pH, which has a detrimental effect on the pathogenic microbes [21]. Moreover, they are also a direct source of energy for enterocytes and cause their hyperplasia, which in turn increase the overall surface of the villi, thus, increasing the absorption of nutrients from the small intestine.

Based on the previous studies, we hypothesized that combined supplementation of O. vulgare, C. verum, citric acid and malic acid (activo^®, EW Nutrition, Niedersachsen, Germany) at different levels and schedules can improve growth performance, immunity and intestinal development. The aim of the current study is, therefore, to investigate the efficacy of supplementation of different concentrations of organic-acid–phytogen composites and to assess their effect on the gut histomorphometry, immune organ development and growth performance in broilers.

2. Materials and Methods

The trial was conducted in an environmental control poultry shed. The cleaning and fumigation of the farm were performed before the arrival of chicks. The litter material comprised rice husks. A total of one hundred forty (n = 140) broilers (Ross 308) were randomly divided into four (4) groups. There were seven (7) replicates in each group containing five (5) broiler chickens each. On day 1, the relative humidity (RH) and temperature were maintained at 65 ± 5% and 35 ± 1 °C, respectively.

From days 1–21, the temperature was reduced on a weekly basis from 3 °C until it reached 26 ± 1 °C. From thereon, relative humidity (65 ± 5%) and temperature (26 ± 1 °C) were maintained until the end of the experiment. Day-old chicks were weighed (g) on arrival, and the subsequent calculation of weight gain was done on a weekly basis and that of feed intake was done on daily basis. The calculated feed consumption and body weight gain were used for feed conversion ratio (FCR) measurement per week. Whereas,

FCR = Feed utilization/body weight gain.

2.1. Study Design

This study was conducted in the University of Veterinary and Animal Sciences, Lahore, Pakistan. The research plan was approved by the Ethical Review Committee for the Use of Laboratory Animals vide Letter No. DR/393 dated 12 April 2019.

The broilers were fed with starter diet (day 1–21) and grower diet (day 22–35) ad libitum (

Table 1. Ingredients in starter (1–21 days) and grower (22–35 days) feed for broilers.

Ingredients (g/kg)	Starter Diet	Grower Diet
Corn	401.0	575.5
Rice broken	150.0	-
Sunflower meal	120.0	130.0
Soya meal	116.4	96.2
Rapeseed meal	50.0	76.0
Molasses	20.0	-
Dicalcium phosphate (DCP)	17.2	19.5
Wheat bran	13.9	-
Rice polish	-	40.0
Canola meal	9.0	50.0
Guar meal	10.0	-
Premix of vitamin mineral *	10.0	10.0
Soda bicarbonate	0.4	0.6
Sodium chloride	2.0	2.0
Composition of Nutrients
Calculated ME (Kcal/Kg)	2750	2850
Dry Matter	870	880
Crude Protein	196.0	185.0
Total Ash	57.5	54.1
Crude Fat	21.7	23.4
Crude Fibre	12.5	18.0

* Premix of vitamin mineral: Ca 195 g, Na 18 g, Mg 6 g, K 70 g, Fe 2000 mg, Mn 1200 mg, Zn 1200 mg, Se 8 mg, I 40 mg, Co 20 mg, Vitamin K 34 mg, thiamine 35 mg, ascorbic acid 1300 mg, riboflavin 135 mg, niacin 1340 mg, folic acid 34 mg, Vitamin B6 100 mg, Vitamin D3 80000 IU, Vitamin A 200000 IU, Vitamin E 1072 IU, biotin 3350 µg, and Vitamin B12 670 µg.

). Activo, a commercial product of organic acids and phytobiotics (activo^®, EW Nutrition, Niedersachsen, Germany) exists in liquid form and consists of citric acid, malic acid, Origanum vulgare and Cinnamomum verum. Birds were divided into four experimental groups. Group-Ι (control group) was fed only on a basal diet (B.D). However, in treatment groups, Group-ΙΙ was fed on B.D + activo (1 mL/3.3 L water for 1–4 days, 1 mL/4 L water from 15–18 days and 1 mL/4 L water from 26–29 days), Group-ΙΙΙ was fed with B.D + activo (1 mL/4 L water from day 1–35), and Group-IV was fed on B.D + activo (1 mL/6 L water from day 1–35).

2.2. Weight and Length of Organs

At the end of the experiment on day 35, two birds/replicate (14 birds/treatment) were selected randomly and were killed through cervical dislocation. Afterwards, the visceral organs (the heart, liver, spleen, bursa of Fabricius, proventiculus, gizzard and intestine) were collected for weighing on a digital weight balance (Shimadzu Corporation, Kyoto, Japan). The lengths of the small and large intestine were measured with a measuring tape.

2.3. Histomorphometry of Gut

Tissue processing was performed through the paraffin embedding technique, followed by hematoxylin and eosin (H&E) staining of the intestinal tissue sections [22]. Two centimeter-long pieces of duodenum, jejunum and ileum were removed. A duodenal segment was selected between the pyloric end of stomach and the distal loop of the duodenum, the jejunum was sampled from between the distal loop of duodenum and the Meckel′s diverticulum, and ileum was sampled from between Meckel′s diverticulum and the ileocecal junction [23].

The intestinal lumen was flushed with normal saline. For histological slide preparation, the sections were placed in 10% neutral buffered formalin (10% NBF). In each cross section of the intestine, five well-developed villi were selected. The selection of villi was based on the presence of intact lamina propria. The villus height (VH) was calculated from the tip of the villus to the junction of villus-crypt. The crypt depth (CD) was measured as the depth between adjacent villi [24].

Based on the VH and CD, the ratio of villus-height-to-crypt-depth was determined. The villus width (VW) was measured from three points: near to the villi tip, the middle of the villi and near to the bottom of villi, and the average of these values was used for statistical analysis. The lamina propria thickness (LPT) was measured between the base of the villi to the muscularis mucosa. The muscularis externa thickness (MET) was also measured. The villus surface area (VSA) was calculated as:

(2 π) × (VW/2) × (VH)

A bright field light microscope (Labomed Inc^®, Los Angeles, CA, USA) was connected with a camera and readings were taken using commercial software (Prog Res^® 2.1.1 Capture Prog Camera Control Software).

2.4. Histomorphometry of Caecal Tonsil and Bursa

Three cross sections were taken from the cecal tonsil and bursa of Fabricius. From each section, one microscopic field under light microscope at 40× magnification was used to measure the lymphatic nodules’ height, width and area in the cecal tonsils. The lymphatic nodule area was calculated using the following formula:

Area of lymphatic nodule: Nodular width × Nodular length.

The bursal lymphoid follicle length, width, area and their total number were also measured, and the area was calculated using the following formula:

Area of lymphatic follicle: Follicular width × Follicular length.

2.5. Histochemistry of Goblet Cells

The goblet cells were stained with combined Alcian Blue and Periodic Acid Schiff (AB-PAS) stain. The goblet cell has an expanded apical portion and a narrow basal portion. Three intestinal tissue sections per sample were examined, and five villi per tissue section were considered for the identification of goblet cells at 100x magnification (Labomed Inc^®, Los Angeles, CA, USA). Goblet cells with neutral mucin, acidic mucin and both acidic and neutral mucin stained magenta, blue and purple respectively [22].

2.6. Intraepithelial Lymphocytes Cells

For the IEL count, slides of intestinal villi were stained with H&E stain and were examined under a light microscope (Labomed Inc^®, Los Angeles, CA, USA) at 400x magnification. Three intestinal tissue sections per sample were analyzed, and five well oriented villi were selected for counting of IEL. Their average was presented as IEL count/villus. Intraepithelial lymphocytes have rounded nuclei surrounded by a narrow cytoplasmic rim [25].

2.7. Connective Tissue Mast Cells

Mast cells were stained with toluidine blue stain. Mast cells contain metachromatic cytoplasmic granules of histamine and heparin [26]. These were quantified in the lamina propria of the small intestine, and five microscopic fields were used for counting [27].

2.8. Statistical Design

The Kolmogorov–Smirnov test was used to determine the normal distribution of data. In SPSS Version 20.0 (IBM, Armonk, NY, USA), and one way analysis of variance (ANOVA) was used to analyze the data. Data are presented as the mean ± S.D. Duncan’s multiple range test was used to compare group differences. The level of significance was kept at p ≤ 0.05.

3. Results

3.1. Growth Traits

The effects of the organic-acid–phytogen composite on healthy broilers are shown in

Table 2. Influence of inclusion of organic acid and phytogen composite on broiler growth performance.

	Grp.Ι	Grp.ΙΙ	Grp.ΙΙΙ	Grp.ΙV	SEM	p-Value
	Weight gain (g)
1st week	120.46	129.30	125.46	128.74	1.70	0.241
2nd week	305.25	310.54	308.68	304.01	3.40	0.910
3rd week	469.55	478.29	494.31	483.00	4.77	0.334
4th week	520.46 b	537.38 a,b	567.74 a	531.80 b	5.96	0.025
5th week	530.19 b	588.58 a,b	612.15 a	576.64 a,b	10.90	0.047
	Feed intake (g)
1st week	144.83	142.61	145.92	143.80	1.05	0.740
2nd week	424.09	420.74	415.98	422.25	3.16	0.846
3rd week	740.31	737.80	730.65	746.09	5.47	0.814
4th week	924.96	936.22	930.27	936.73	5.50	0.832
5th week	1093.35	1079.21	1064.96	1085.84	6.85	0.531
	FCR (gram feed/gram body weight gain)
1st week	1.21±	1.10	1.16	1.12	0.01	0.156
2nd week	1.39±	1.35	1.35	1.39	0.01	0.749
3rd week	1.57±	1.54	1.47	1.54	0.01	0.183
4th week	1.78 a	1.74 a	1.64 b	1.76 a	0.01	0.045
5th week	2.06 a	1.84 b	1.74 b	1.90 ab	0.03	0.026

^a,b Means with different superscripts (within the same row) are differ significantly (p ≤ 0.05). Values represent the Mean ± SEM of seven replicates. Grp: group. Group-Ι: Control group. Group-ΙΙ: B.D + activo 1 mL/3.3 L water for 1–4 days, 1 mL/4 L water from 15–18 days and 1 mL/4 L water from 26–29 days. Group-ΙΙΙ: B.D + activo 1 mL/4 L water from day 1–35. Group-ΙV: B.D + activo 1 mL/6 L water from days 1–35.

and

Table 3. Influence of inclusion of the organic acid and phytogen composite on the broiler absolute organ weight (g)/ length (cm).

	Grp.Ι	Grp.ΙΙ	Grp.ΙΙΙ	Grp.ΙV	SEM	p-Value
	Organ weight
Bursa (g)	0.920	0.958	0.945	0.970	0.007	0.086
Spleen (g)	1.707	1.671	1.664	1.678	0.020	0.890
Heart (g)	10.92	11.64	12.28	11.28	0.236	0.214
Liver (g)	43.0	42.35	43.428	41.71	0.247	0.072
Proventiculus_F (g)	7.987	8.17	8.23	8.21	0.100	0.791
Proventiculus_E (g)	4.32	4.47	4.48	4.38	0.082	0.888
Gizzard_F (g)	38.71	40.42	40.64	39.28	0.529	0.529
Gizzard_E (g)	26.57	27.92	28.28	27.57	0.254	0.094
Small Intestine_F (g)	86.78	88.64	90.14	87.14	1.081	0.690
Small Intestine _E (g)	57.92	59.78	61.07	58.07	0.984	0.640
Cecum_F (g)	7.19	7.74	8.09	7.42	0.127	0.066
Cecum_E (g)	4.98	5.43	5.63	5.17	0.094	0.071
Small Intestine_L (cm)	177.85	180.71	184.42	181.85	1.229	0.304
Cecum_L (cm)	9.97 c	10.18 a,b	10.35 a	10.11 b,c	0.039	0.004

^a–c Means with different superscripts (within the same row) are differ significantly (p < 0.05). Values represent the Mean ± SEM of seven replicates. Group-Ι: Control group. Group-ΙΙ: B.D + activo 1 mL/3.3 L water for 1–4 days, 1 mL/4 L water from 15–18 days and 1 mL/4 L water from 26–29 days. Group-ΙΙΙ: B.D + activo 1 mL/4 L water from day 1–35. Group-ΙV: B.D + activo 1 mL/6 L water from day 1–35. E = empty, F = filled, and L = length.

. The feed intake of all treatment groups was the same (p > 0.05). The FCR during the fourth week of group III was lower (p ≤ 0.05) than the control group and other supplemented groups. During the fifth week, the FCR of group III and IV was lower (p ≤ 0.05) than the control group. However, the weight gain of group III was higher (p ≤ 0.05) than the control group during the fourth and fifth weeks.

The weights and lengths of organs did not vary among groups (p > 0.05), except the cecal length, in which group III was significantly higher (p ≤ 0.05) as compared to the control (Table 3).

3.2. Gut Histomorphometry

The effect of the organic-acid–phytogen composite on broiler gut morphometry is shown in Figure 1 and labelled in the Figure 3a. The gut histomorphometric parameters showed that the VH and VH/CD ratio of all intestinal segments were higher (p ≤ 0.05) in group III as compared to the control. The VSA was higher (p ≤ 0.05) in duodenum and jejunum in group III compared with the control. The thickness of the muscularis externa was greater (p ≤ 0.05) in the ileum of the control group when compared with group IV.

3.3. Morphometry of Immune Organs

The effect of the organic-acid–phytogen composite on the mormphometry of the immune organs of broilers is shown in Figure 2. The lymphatic follicular number in the bursa of Fabricius was higher in the control group (p ≤ 0.05) when compared to other supplemented groups and labelled in the Figure 3d. The lymphatic follicular width and area was greater in group III and IV compared with the control. The lymphatic nodular length and width in cecal tonsils were the highest (p ≤ 0.05) in group III and labelled in the Figure 3e.

The number of acidic goblet cells was higher in group II in all segments (duodenal, jejunal and ileal) of the small intestine in comparison with the control group. In the duodenum, mixed goblet cell count was higher (p ≤ 0.05) in group II compared to the control and group IV. In the duodenum, total goblet cell count was higher (p ≤ 0.05) in group II when compared to the control and group III. In jejunum, total goblet cell count was higher (p ≤ 0.05) in all supplemented groups when compared with the control and in the ileum, the count was higher (p ≤ 0.05) in group II and III in comparison to the control. As labelled in Figure 3c.

In the duodenum and jejunum, the intraepithelial lymphocyte number was similar (p > 0.05) among all groups and labelled in the Figure 3b. In the ileum, the IEL count was greater (p ≤ 0.05) in the control group as compared to group-ΙΙΙ. The mast cell count did not vary among the groups.

4. Discussion

Organic acid and phytogen are highly potent growth promoters as well as antimicrobial and immunomodulatory compounds for poultry birds. Various studies have reported their individual use as alternatives to antibiotics in poultry feed. However, their combined use is still not reported, particularly that of organic acid (citric acid and malic acid) in combination with phytogen (Origanum vulgare and Cinnamomum verum) in drinking water.

To the best of our knowledge, this is the first scientific study that reports the combined use of organic acid (citric acid and malic acid) and phytogen (Origanum vulgare and Cinnamomum verum) as a growth promoter in broilers. In the present study, during the fourth and fifth weeks of the experiment, organic acid and phytogen supplementation in the water had a positive effect (p ≤ 0.05) on growth performance (weight gain and FCR).

Our findings concurred previous ones, which reported that, during the fourth and fifth weeks, the combined essential oil and organic acid blend improved weight gain and FCR [28]. Organic acid and essential oils in combination have been reported to decrease pathogenic bacteria in the intestine of poultry [29,30] due to lowering the gut pH [31]. This may function to limit the damage to intestinal villi and thereby support the absorption of nutrients to improve body weight gain. Contrarily, [32], observed that there was no significant impact of organic acid and phytobiotics on growth performance in broilers. The difference in our results may be due to the breed difference of animals (they used Cobb 500) and supplement composition of organic acid and phytobiotic as these are microencapsulated products.

Villus surface area (VSA) and VH/CD ratio, which are the main indicators of absorption and digestion capability of the intestinal villi are linked to intestinal integrity and health. In the current study, VH and VH:CD in each segment of the small intestine and villus surface area in the duodenum and jejunum were significantly higher in birds supplemented with organic acid and phytogen composite. Our results agree with previous studies, where a composite supplementation regime of organic acids and phytogen improved the VH, VSA and VH:CD [33,34].

Dietary organic acids cause enterocyte hyperplasia in the intestinal mucosa, which leads to an increase in nutrient absorption, accelerates bird growth and improves their antioxidant status. The antioxidative effects of organic acid increase the VH, which may delay apoptosis and increase enterocyte viability [35]. Oregano and cinnamon are lipophilic in nature and easily pass through the bacterial cell membrane, which disrupts the homeostasis of bacteria due to lowering their pH [36].

This ultimately leads to the death of pathogenic bacteria, thus, decreasing or halting the colonization of pathogenic bacteria in the intestine [37]. This in turn enhances the gut health via development of the villus height and surface area. Increase in the villus surface area has positive effects on the mucosal enzymes as well as absorption and digestion.

In the current study, the supplementation of organic acid in combination with phytogen improved the morphology of immune organs and potentially improved the immunity against pathogens, in broilers. Moreover, the lymphatic follicular number of the bursa of Fabricius was lower in group III as compared to the control, and their width and area were higher in group III. In cecal tonsils, the lymphatic nodular length and width in group-ΙΙΙ was higher.

Together, these observations indicate that organic acid in combination with phytogen may have a role in improving broiler immunity. Our results are in accordance with a previous study that observed that organic acid supplementation in the diet enhanced the structure of the lymphoid follicles and indicated an improvement in the immune status of poultry [38].

These bursal follicles contain lymphoid cells, which are directly related to the immune status of birds [39]. Organic acids stimulate the natural immune system, which in turn kills the pathogenic bacteria in the gut flora of poultry [40]. Moreover, their supplementation stimulates immune response, which is then observed as an increase in globulin serum levels against pathogens in poultry [41].

The number of goblet cells in the intestine mucosa reflects the intestinal capacity for mucin production. Goblet cells are, therefore, a component of the gut immune system. Mucin is glycoprotein in nature and present on the superficial layer of the broiler gut mucosa. It functions to lubricate the intestinal surface and to identify and neutralize the pathogenic bacteria.

Moreover, it helps in the digestion and absorption of nutrients and detoxification of heavy metals [42]. We observed that the number of both acidic and total goblet cells in each segment of the small intestine was higher in group II as compared to the control. Similarly [43], reported that the supplementation of organic acid resulted in higher acidic goblet cells number in broiler jejunum.

Acidic mucin has the potential of protecting against pathogenic microbiota due to resistance against harmful action of bacterial proteases [44]. Intraepithelial lymphocytes (IEL) are T-cells that are present in spaces of intestinal epithelium. They are primary defenders against entero-pathogens [45]. The IEL count is subject to change in situations, such as dietary changes, the appearance of diseases and even during other management changes. In the current study, control group ileal villi had a lower (p ≤ 0.05) number of IEL than the treatment groups.

The reason for the lower number of IEL counts could be that thymol (active ingredient of oregano) decreases the formation of prostaglandins by inhibiting cyclooxygenase. Prostaglandins are mediators of inflammation. Thus, due to a controlled inflammatory response, the flow of T-lymphocytes towards the intestinal mucosa reduces [46]. Cinnamaldehyde also has anti-inflammatory properties as it make changes in the mRNAs levels related to 62 genes [47]. Therefore, a decrease in the IEL count in the small intestine indicates better mucosal immunity.

5. Conclusions

The supplementation of organic acid (citric acid and malic acid) in combination with phytogen (Origanum vulgare and Cinnamomum verum) in drinking water improved growth performance through the enhancement of intestinal histomorphometry. It also improved the morphology of immune organs i.e the bursa of fabricius, cecal tonsils, goblet cells and intra epithelial lymphocytes in broilers. The organic acid phytogen composite improved the gut health by decreasing the load of pathogenic bacteria.