Current Research in Poultry Science

Year: 2021 | Volume: 11 | Issue: 1 | Page No.: 1-10
DOI: 10.17311/crpsaj.2021.1.10

Synergistic Effects of Synbiotics and Essential Oil on Coccidian Control in Broiler Chickens

M. Eldeib , S.A. El Safty and M.M. Hashim

Abstract: Background and Objective: The use of antibiotic growth promoters and coccidiostats drugs in poultry feed led to human health hazard, it has become necessary to develop a natural alternative. This study discovers the possible synergistic effect of probiotics, prebiotics, butyrate and essential oil (All-Pro feed) that can be beneficial for improving the growth performance of broiler chickens and coccidian control. Materials and Methods: A total of 480 one-day-old Ross 308 broiler chicks (as hatched) were subjected to a 5 weeks dietary experiment. The chicks were randomly divided into 2 experimental groups, group 1 fed basal diets only (T₁), group 2 fed basal diets supplemented with 750 g t^–1 (All-Pro feed) T₂. Each treatment comprised 4 replicates (60 chicks per replicate). It includes parameters for production performance as body weight gain feed consumed and feed conversion ratio. Immunological, histomorphology and coccidian study. Statistical analysis was conducted using the student’s t-test. Results: There was a significant increase in Body Weight Gain (BWG) in the group treated with All-Pro feed compared with the control group in the hall period of the experiment. There was a significant improvement in Feed Conversion Ratio (FCR) between the control and treated group except for the 3rd week. Adding All-Pro feed increased villus height and width linearly, increased humoral immunity and reduced coccidian oocyte shedding in faeces of chickens at different ages. Conclusion: From the above study can prove the synergistic action of herbal extract with synbiotic to control coccidiosis in chickens in addition to its role as growth promoters.

Keywords: Probiotics, prebiotics, butyrate, essential oil, histomorphology, coccidiosis and immunity

INTRODUCTION

Growth promoters are chemical and biological substances that are added to livestock feed to improve the utilization of feed and in this way realize better production and financial results. Antibiotics have been used as growth promoters for decades¹. However, the use of most antibiotic growth promoters has been banned in many countries because of public concerns about their residues in animal products and the development of antibiotic-resistant bacteria². As a consequence, it has become necessary to develop alternatives using either beneficial microorganisms (probiotics) or non-digestible ingredients (prebiotics) that enhance microbial growth. A way of potentiating the efficacy of probiotics preparation may be the combination of both probiotics and prebiotics (synbiotic) that beneficially affects the host by improving the survival and implantation of live dietary microbial supplements in the gastrointestinal tract. Those effects are due to activating the metabolism of one or a limited number of health-promoting bacteria or by selectively stimulating their growth, which improved the welfare of the host or both³.

Phylogenic considered one of the promising alternatives because of its significant potentiality to promote growth, enhance feed intake and stimulate immunity⁴. Besides, they could improve feed intake, feed conversion ratio and carcass yield⁵.

Butyrate is important as food for cells lining the colon (colonocytes). Without butyrate for energy, colon cells undergo autophagy (self-digestion) and die⁶. Short Chain Fatty Acids (SCFAs), which include butyrate are produced by beneficial colonic bacteria (probiotics) that feed on or ferment prebiotics, which are plant products that contain adequate amounts of dietary fibre. These SCFAs benefit the colonocytes (cells of the colon) by increasing energy production and cell proliferation. When butyrate is present in the bloodstream or the proximal parts of the intestinal tract, it induces the production of host defence peptides⁷. These peptides stimulate the development and repair of the intestinal tract through an increase in cell proliferation⁸. Recently it has been shown that butyrate, when present in the blood, stimulates a peptide that increases the absorption of glucose from the intestine. Indications that a similar mode of action can be expected in poultry, is shown by previous study⁹, which found an increased development of the villi when sodium-butyrate was added to the diet. Butyrate also has been shown to stimulate several functions in the lower part of the intestinal tract. Studies have identified specific G-protein-coupled receptors, specifically G-Protein-Coupled Receptor 41 (GPR 41) and GPR 43, on gut epithelial cells in the epithelium of particularly the ileum, caeca and colon¹⁰. When butyrate is attached to these receptors the production of several different peptides is stimulated^11,12. Some of these peptides have a positive effect on the development of the immune system and improve the functioning of the immune system in case of a health challenge¹¹. Other peptides have been shown to optimize gut motility, by reducing the rate of feed passage¹. In poultry, the emptying of the feed out of the gizzard into the small intestine is slowed down. Thus, it seems that butyrate is inducing a similar effect to passage rate as coarse particles such as an oyster shell.

Indications that butyrate also stimulates the immune system in poultry were obtained by Leeson et al.¹³ in that, birds previously fed butyrate showed more ability to withstand the stress of coccidial challenge at 21 days of age. Weber and Kesr¹⁴ found that when pigs were challenged with Escherichia coli Lipopolysaccharide (LPS), sodium butyrate increased the magnitude of the cortisol response and increased skeletal muscle IL-6 mRNA expression, also indicating that dietary butyrate affects the response to inflammatory stimuli.

The objectives of the current study were to evaluate the effects of a mixture of prebiotics, probiotics, butyrate and herbal extracts (All-Pro feed^®) on growth performance, small intestine histopathology, coccidian oocytes excretion and humoral immunity.

MATERIALS AND METHODS

Study area: This trial was carried out at Poultry Production Farm, Feed Research House, Orabi Community, Qalyobia Governorate Egypt during 2019-2020. The laboratories study was carried out at the Animal Health Research Institute of Pharmacology and Parasitology Laboratory, Giza, Egypt.

Experimentation: A total of 480 Ross 308 broiler chicks (as hatched) at one-day-old with an initial body weight of around 44 g were obtained from a local commercial hatchery. The chicks were weighed and randomly distributed in 8 pens to examine the effect of 2 experimental treatments comprising two feeding regimens: (T₁) Basal diet without All-Pro feed supplement (control group) and a basal diet supplemented with 750 g t^–1 All-Pro feed (T₂).

Each group contained 240 chicks which were allotted into 4 replicates and each replicate contained 60 chicks. All the experimental birds were reared in a well-ventilated shed on softwood shaving litter used as bedding material in pens with 2.0×3.0 m² dimensions and kept under uniform management conditions.

Basal starter (0-14 days), grower (15-24 days) and finisher (25 day-end) diets were formulated according to the nutritional recommendation for broilers¹⁵, their composition and calculated analysis are shown in Table 1. All-Pro feed Powder is a dry stabilized preparation manufactured by (Kanzy Medipharm, Canada) it is a mixture of probiotics, prebiotics, butyrate, essential oil and silicate as (carrier). Feed and water were available ad-libitum during the experiment. All the birds were weighed at the same time weekly before feeding.

Feed intake calculation: Feed intake was calculated by measuring the amount of feed offered and residue left. The Feed Conversion Ratio (FCR) was calculated by dividing the feed intake by weight gain.

Parameters for productive performance (body weight, body weight gain, feed consumption and feed conversion ratio) were measured on weekly basis throughout the experimental period. The body weight gain of broilers was measured as a difference of weight between two weighing intervals.

After correction for feed refusals, feed consumption was calculated using the following formula:

Feed conversion ratio = feed consumed in each replicate/total weight gain (with a weight gain of died or culled chickens).

Vaccination and other routine poultry management practices were carried out neatly.

The groups were experimentally infected with coccidiosis. For this purpose, a suspension containing E. tenella oocytes in 2.5% potassium dichromate solution a 100,000 oocysts per 1 mL suspension. The groups of chickens were infected for 14 days by giving 1 mL suspension containing E. tenella oocytes by gavages.

Histopathological study: For histopathology, slides were prepared for duodenum samples of the dissected birds on day 17, 24 and 32 of age as the duodenum was considered the part from loop along with the pancreas up to bile ducts insertion. Samples were cleaned with distilled water to remove the intestinal contents and fixed in 10% neutral buffered formalin solution then dehydrated, cleared and embedded in paraffin wax, then specimens were sectioned to 4-5 micron thickness, prepared and stained with Hematoxylin and Eosin stain (H and E) and examined microscopically. Morphometric measurements of duodenum villi were performed including villus length and villus width the height of the intestinal villi was determined by measuring from the base of the lamina propria to the tip of the villi, for duodenum 9-10 intestinal villi per sample were measured for each intestinal section and each bird was measured at 40 magnifications using ImageJ Analyzer software¹⁶.

Oocyst counting: A sample of 1 g of rectal content was taken from each specimen at 17, 24 and 32 days of age, then mixed with 10 mL physiological saline, sieved in a 150 micro-mesh to remove debris and poured in a test tube then centrifuge at 1500 rpm for 5 min. Heavy material in the dropping stetted at the bottom and any suspension was thrown away. The material that remained in the test tube was mixed with 10 mL Aluminum Nitrate and samples are removed with a Pasteur pipette and a McMaster counting chamber is filled. The number of oocysts was counted under a microscope (Long et al.¹⁷).

where, n is number of oocysts counted, Vol. = 10 mL physiological saline that faeces is soaked in, 0.15 volume of the Mc-Master counting chamber.

Immunity assessment: To investigate the possible effect of All-Pro feed on humoral immunity, an immunoassay was adopted. For this purpose, blood samples were collected from wing veins from 5 randomly selected birds at weekly intervals (1-5 weeks of age) from each group. The serum samples were subjected to HI test for determining antibody titers against Newcastle Disease (ND) vaccine employing 8 HA units as described by Swayne et al.¹⁸.

Statistical analysis: Statistical analysis was conducted using the student’s t-test¹⁹.

RESULTS AND DISCUSSION

The live body weight and average weight gain of broiler as affected by dietary treatments are illustrated in Table 2. Results revealed that chickens fed diets supplemented with All-Pro feed Powder (T₂) exhibited significant (p<0.05) heavier live body weight (2098±29.48) at marketing age than chickens fed the control diet (1983.51±19.44).

The results indicated a significant difference due to the main effects of dietary treatments on Body Weight Gain (BWG)during the breeding period.

These results are in the same line with the results published by Ocak et al.²⁰, who reported significantly higher LBW at 21 and 42 days of ages as well as higher BWG from 7-35 days of age in broilers fed peppermint and thyme compared to the control group. Also, Al-Kassie²¹ and Abid²² showed that chickens fed with thyme supplement had significantly heavier live body weight and body weight gain compared with those fed the control diet. Another study showed that an addition of 5 g kg^–1 thyme herb improved BWG by about 6% when compared to the corresponding control group²³. On the contrary, other studies showed that using 5 g kg^–1 thyme caused a substantial decrease in BWG approaching almost a level of significance²⁴.

Studies on the beneficial impact on chicken's performance have indicated that probiotic supplementation can have positive effects. It is evident from the result of Kabir et al.²⁵ that the live weight gains were significantly (p<0.01) higher in the experimental birds as compared to control ones during the period of 2nd, 4th, 5th and 6th weeks of age. Besides Torres-Rodriguez et al.²⁶ reported that administration of the selected probiotic (FM-B11) to turkeys increased the average daily gain and marked body weight (BIV) representing an economic alternative to improve turkey production. Mechanisms by which probiotics improve feed conversion efficiency include alteration in the intestinal flora, enhancement of growth of nonpathogenic facultative anaerobic and gram-positive bacteria forming lactic acid and hydrogen peroxide, suppression of growth of intestinal pathogens and enhancement of digestion and utilization of nutrients²⁷. Using probiotic microorganisms shorten the period needed to stabilize the microflora. This microflora regulation may serve to improve feed conversion, weight gain and also improve the intestinal health and immune competence of the chickens²⁸. For feed consumption trait, it could be observed that during the studied period (0-5 weeks), there was no statistically significant difference among treatments in feed consumption trait except starter period there was a mild significant difference between T₂ (441.41±4.31) and T₁ (426.23± 2.40). Accordingly, Lee et al.²⁹ the positive effects can be evaluated based on different perspectives as thyme improves diet palatability by influencing the main components of the thymus that stimulate and improve the appetite and the digestive process. The Feed Conversion Ratio (FCR) describes the relationship between feed intake and Body Weight (BW) gain. More precisely, it is the animal’s overall efficiency in converting feed mass into body mass over a specific time. It was obvious from Table 2 that during starter or grower periods, there was a significant effect on FCR among treatments except for 3rd week. Whereas, during the finisher period, chickens fed (T₂) diet were more efficient (1.90±0.15) in converting their feed into body weight gain compared with control group T₁ (2.14±0.05).

Accordingly, the improved FCR could be due to enhancing the efficiency of energy and nutrient utilization or altered carcass composition³⁰. On the other hand, Alagawany et al.³¹ and Zhang et al.³² found that supplementation of broiler diet with thyme had a significant positive effect on the FCR of broilers. However, several commercial herbal blends were included in trials but only two studies confirmed a significant improvement of FCR. The addition of 100 mg kg^–1 of RepaXol^® optimized FCR by 4%³³. Furthermore, using oregano herbs tested in broiler diets improved body weight and feed intake at 1,000³⁴, 500³⁵, 100 and 250 ppm³⁶. Global financial losses due to coccidiosis in broiler flocks are estimated to be more than 3 billion annually³⁷. The addition of coccidiostats to poultry feed can control the diseases but the emergence of drug resistance as well as possible future ban restricting coccidiostat use mean that there is an urgent need for an alternative method of reducing the burden of disease in poultry³⁸. Effect of All-Pro feed on oocytes count at different ages (21 and 32 days of age) T₂, comparing with the control groups T₁ was obvious in Table 3. All-Pro feed treated groups had a significant decrease in the number of shaded oocysts after 21 days (454,000±38807) compared to the T₁ group (698,000±33643). While there was a highly significant difference between T₁ (822,200±32456) and T₂ (322400 ±33643) on day 32. Essential oils contain known active ingredients including, phenols, aldehydes, terpenes and oxides which have a direct anti-parasitic effect. The majority of the antimicrobial prowess of carvacrol and thymol can be attributed to creates membrane permeability problems for the organisms³⁹. Carvacrol has been shown to activate and desensitize receptors in calcium channels⁴⁰. Carvacrol may to the observed inhibition of sporozoites invasion by disrupting calcium-mediated signalling in the sporozoites. Gianmenas et al.⁴¹ reported that carvacrol and thymol increased broilers weight gain and lower oocyst counts compared to infected, untreated controls. Finally, Ovideo-Rondon et al.⁴² showed that treatment of Eimeria spp. infected broilers with two specific EO blends (containing thymol, eugenol, curcumin, piperine) prevented major shifts in the intestinal microbial communities as compared to non-treated controls, leading to less severe clinical signs.

The results of these studies suggested that in broilers, essential oils could be an alternative to drugs and/or vaccination for protection against E. tenella infection. The results of the humoral immune response are presented in Table 4 which indicated a significant increase in the All-Pro feed treated group (7.86±0.121) over the control group (6.9±0.071) in the HI titers against ND vaccine at different examined intervals. Brander et al.⁴³ reported that immunomodulators administered simultaneously with antigens might potentiate specific immune responses, particularly to vaccines. Chau et al.⁴⁴ concluded that β-Glucan can be efficacious as an oral adjuvant to enhance immunoglobulin production in response to vaccination. Williams et al.⁴⁵ and Lowry et al.⁴⁶ suggested that the protective effect of β-glucans might be due to the antioxidant capacity, antimicrobial activities as well as the inhibition of early activation of tissue muscular Nuclear Factor-KB (NF-KB) and NF-IL6. In the present investigation, the results of villus height and width of the control and treated groups are given in Table 5. Findings of the intestinal villi to the control group fed basal diet showing normal length with lining epithelial simple columnar absorptive cell and goblet cells with slightly increasing of the average length of the villi with times after slaughter 17, 24 and 32 days by scale bar measurement 495.4±33.65, 548.2±4.71 and 582.6±19.14 respectively and also slightly increasing of the average width of the villi with times after slaughter 17, 24 and 32 days by scale bar measurement 83.4±4.40, 84.4±3.58 and 87.1±7.46 um respectively. The luminal side of the intestinal wall is lined with absorptive columnar epithelial cells, needed for water and nutrient uptake and mucin-producing goblet cells which are important in innate defences that form a semi-permeable barrier. This semi-permeable formed by the cell membranes of the epithelial cells and tight junctions that connect neighbouring epithelial cells⁴⁷. The permeability of the intestinal epithelial cell layer can be affected by epithelial cell death and also by luminal signals that increase the epithelial layer permeability by affecting the tight junctions and thus cause loss of integrity of this important barrier⁴⁸. Regarding intestinal histomorphology of the chickens in the treated group fed basal diet and 750 g t^–1 of All-Pro feed showing increasing the length of the intestinal villi with increasing the time after slaughter 17, 24 and 32 days with average length 655.6±1, 698.8±1 and 811.2±1 respectively and average width 80.2±1, 81.1±1 and 65.3±1 respectively. The villus height was significantly longer in the T₂ group which was given All-Pro feed in a dose of 750 g t^–1, besides increasing the length of villi with age, so villi were longer at day 32 than 24 and 17 days old of chickens, respectively. However, villus width did not differ significantly in the experimental groups as well as in the control. These results may be due to higher levels of intestinal probiotics in birds fed with Mannan Oligosaccharides (MOS) that resulted in the improvement of gut health status. Mannan oligosaccharides have been reported to increase villus height, surface area and decrease crypt depth^49,50. Similarly, Cheled-Shoval et al.⁵¹ reported that administration of MOS enhanced the villus area. When butyrate is present in the bloodstream or the proximal parts of the intestinal tract it induces the production of host defence peptides⁷ these peptides stimulate the development and repair of the intestinal tract through an increase in cell proliferation⁸ recently it has been shown that butyrate, when present in the blood, stimulates a peptide that increases the absorption of glucose from the intestine. Hu and Guo⁹ found an increased development of the villi when sodium butyrate was added to the diet.

When butyrate is attached to specific G-protein- coupled receptors specifically GPR 41 and GPR 43 on ileum epithelial cells, the production of several different peptides is stimulated, some of these peptides have a positive effect on the development of the immune system¹¹ other peptides have been shown to optimize gut motility, by reducing the rate of feed passage¹². Also, Alfaro et al.⁵² and Alaeldein et al.⁵³ mentioned that chickens fed phytogenics feed additives were showed longer ileal villi with excellent gut health, high absorptive efficiency and healthier intestinal tract. The recent studies reported that dietary supplementation of thymol and carvacrol reduced intestinal lesions, improved the intestinal histomorphology and enhanced the specific immune response⁵⁴. In the present study chickens treated with basal diets supplemented with 750 g t^–1 (All-Pro feed) in the 2nd group showed proliferation in glandular lining epithelial cells of the intestinal villi (Fig. 1) and simple columnar epithelium containing hyperplasia of goblet cells and the core of the villi containing loose connective tissue in 2nd group (Fig. 2). These results are agreed with the previous researchers^55-57 as they mentioned that Mannan oligosaccharides have been reported to induce numbers of sulphated-acidic goblet cells. It has been reported that sulphated-acidic goblet cells are less degradable by the pathogen's glycosides^58,59. Therefore, they can provide stronger protection against pathogens for the host. Similarly, Cheled et al.⁵¹ reported that administration of MOS enhanced the proliferation of goblet cells, synthesizing and secreting more mucin, which plays an important role as the first line of defence. Mucin can trap pathogens or impede them from invading epithelial cells⁶⁰. Besides, higher levels of intestinal prebiotics and probiotics in birds feed may further result in the improvement of gut health status, the increase of probiotics may improve intestinal development, whereas mucin produced by goblet cells can conversely limit attachment of pathogens to epithelial cells and prevent its invasion so improving health and productivity of broilers⁶¹. Also, Supplementation of butyrate in the feed can beneficially influence growth performance and intestinal villus structure in broiler chickens⁴⁹ which play role in the control of pathogens such as Salmonella enteritidis and Clostridium perfringens^62,63 as well as a decrease of necrotic lesions induced by C. perfringens in the small intestine⁶⁴. Generally, when butyrate-producing bacteria are present in a sufficiently high concentration, the epithelial barrier integrity will be stronger, the epithelial cells will proliferate more and thus the villi will be longer. Moreover, inflammatory reactions will be reduced, while the stimulation of regulatory T-lymphocytes will yield a state of tolerance toward non-harmful bacteria⁶⁵. The present finding suggested using butyrate with synbiotics to improve body weight gain and use essential oil in the control of coccidian in the recommended dose, above dose may cause a negative result and need to study in another research article.

CONCLUSION

The present finding provides evidence of the encouraging role of All-Pro feed as coccidian control in chickens without using anti-coccidian drugs. Besides its role in improving bird growth performance and intestinal histomorphology. More studies are needed to explore its capabilities to support this study.

SIGNIFICANCE STATEMENT

This study discovered the possible synergistic effects of symbiotic, butyrate and essential oil combination to control coccidiosis and improve growth rate in broiler chickens. This study will help the researcher to uncover the critical area of how butyrate works in GIT and improve the histomorphology picture of the intestine that many researchers were not able to explore.

REFERENCES