Effects of in Ovo Infusion of Probiotic Strains on Performance Parameters, Jejunal Bacterial Population and Mucin Gene Expression in Broiler Chicken

ABSTRACT

The objective of the present study was to evaluate the effects of in ovo infusion of probiotic strains (Bacillus subtilis, Enterococcus faecium, and Pediococcus acidilactici ) on jejunal microbial population and mucin gene expression in broiler chicken. In a completely randomized design, 0.5 ml of mediums containing 10⁷ cfu of different probiotic strains, was administered into amniotic fluid of the 480 Cobb fetus (day 18 of incubation), with four treatments, five replicates with twenty four eggs each. For mucin gene expression, samples from the jejunum were taken on day 21 of incubation and day 3 post-hatch. Microbial profile was determined for total lactobacillus and E. coli by sampling jejunal contents on days 1 and 3 of age. Expression of the mucin gene in the jejunum was higher (p<0.05) in chicks that received Bacillus subtilis in comparison with the control group. Infusion of the probiotic strains had no effect on jejunal E. coli and lactic acid bacteria populations on day 1 post-hatch (p>0.05). There were no significant differences among treatments for performance parameters at different periods and the whole period. It was concluded that infusion of probiotic bacteria during the late of incubation has no effect on feed intake, gain and feed conversion ratio, but has a positive effect on mucin gene expression in the jejunum. The best probiotic strain for mucin gene expression was Bacillus subtilis and for beneficiary bacteria colonization was Bacillus subtilis and Pediococcus acidilactici .

Keywords:
In ovo infusion; Jejunum; Microbial population; qPCR; Probiotic

INTRODUCTION

The application of probiotics as an alternative of antibiotics in non-ruminant rations has become necessary. Crawford (1979) reported that probiotics could increase the population of beneficial bacteria and reduce the growth of pathogenic flora in the gastrointestinal tract. Fuller (1989) concluded that probiotics are suitable alternatives for antibiotics as the latter have residues in meat, increase the resistant bacteria and resulted in imbalance of normal microflora. The action modes of probiotics, which affect gut function and health in poultry, include maintaining a normal micro-flora and beneficial microbial population by competitive exclusion and antagonism (Fuller, 1989), and improving gut mucin composition and amount (Tsirtsikos et al., 2012).

Modern poultry production excludes the contact between chick and the hen. Sterzo et al., (2005) reported that bacteria present in the hatchery determine the colonization of beneficial or pathogenic bacteria. Cukrowska et al. (2002) showed that pathogens can be lodged in the chick´s intestine and colonize in the first contact with microbes in the hatchery, so it is necessary to inoculate probiotic bacteria before the chick and hen can be in contact.

The majority of gastrointestinal tract is covered by a viscoelastic mucous gel layer that acts as a protective barrier against potential physical and chemical hazards in the luminal environment (Dharmani et al., 2009). The mucins as main component of the mucus layer, are produced and secreted by goblet cells. Development of the small intestinal mucus-secreting cells in chicks occurs in the late embryonic and immediate post-hatch period. The Mucin-producing cells were present in the small intestine with no differentiation between various sections from 17 days of incubation and produce only acidic mucin (Uni et al., 2003). After hatching, an increase in the goblet cells density production, neutral and acidic mucin was observed from duodenal to ileal axis (Uni et al., 2003).

Fuller (1989) and Tsirtsikos et al. (2012) reported potential link between gut mucin composition and gut microflora described earlier in broiler chicks. Uni & Ferket (2004) and Tako et al. (2004) reported that intra-amniotic nutrient injection accelerated small intestine development and had an enhanced effect on the function of enterocytes.

In the poultry industry, exclusion of the contact between chick and the hen causes a delay in gut colonization with desirable microorganisms. This exclusion exposes chicks to risk of colonization with pathogenic bacteria present in the hatchery (Cukrowska et al., 2002). Thus, injection of probiotic bacteria intra egg may be an alternative to microbiota acquisition by chicks before hatching and may reduce or avoid the gastrointestinal colonization by pathogens. Three species of bacteria, Bacillus subtilis, Enterococcus faecium, and Pediococcus acidilactici , are naturally occurring microbiota in the intestine of birds and common in commercial probiotic products. Information concerning the effects of in ovo infusion of these probiotics on beneficial and pathogenic bacterial population and mucin gene expression in the intestine of chicks, is limited. Therefore, the main objective of this study was to evaluate the effects of different probiotic strains as in ovo infusion on the performance parameters, mucin gene expression and bacterial population in the jejunum of broiler chicken at the pre and post-hatch periods.

MATERIAL AND METHODS

Fertile Cobb chicken eggs were obtained from a commercial hatchery from 38 weeks old breeding flock. Eggs were incubated in single-stage incubators under the same condition of 37.6 °C and 60% relative humidity, while being turned once per hour. On day 17 of incubation, eggs with live fetus (no: 480, average weight of 58 ± 1.1 g) were selected and weighed. In a completely randomized design, eggs were assigned to four experimental groups with five replicates of each twenty four eggs. Infusion of probiotic strains was done for 3 hours in a plastic tent constructed in front of setter doors, with two heaters at 36 °C and 55% relative humidity. The four treatment groups that received in ovo 0.5 ml of sterile distilled water or probiotic mediums (10⁷ cfu) into the amniotic fluid were: 1) sterile distilled water as control group, 2) Bacillus subtilis , 3), Enterococcus faecium and 4) Pediococcus acidilactici . Bacillus subtilis was obtained from Iranian TaqGen Company (Tehran, Iran), Enterococcus faecium was obtained from Biochem Company ( Lohne, Germany) and Pediococcus acidilactici was obtained from Lallemand Inc. (Paris, France).

The in ovo infusion procedure was performed as described by Tako et al. (2004). Solution was injected with a suitable needle inserted into the amniotic fluid, which was identified by candling. After injection, the hole in the egg shell was sealed with cellophane tape, and eggs were placed in hatching trays. Chicks were raised for 42 days and fed Cobb standard rations (Table 1). Chickens management (water, feed, bed material, light program and pen environment) were the same in all groups and were based on Cobb 500 broiler chickens (Cobb Manual Guide, 2012). Chickens were weighed in the final of starter (day 10) and finisher (day 42) to estimate their growth. To determine daily feed intake by each pen, the uneaten feed was discarded and fresh feed was replaced in feeders at the end of each day. Feed conversion ratio was calculated in the mentioned periods.

Table 1
Ingredients and compositions of rations

Intake period			Ingredients (%)
Finisher (days 29-42)	Grower (days 11-28)	Starter (days 1-10)
62.59	60.95	55.06	Corn grain
29.13	31.52	37.05	Soybean meal (46% CP)
4.35	3.50	3.51	Soybean oil
0.92	0.91	0.95	Calcium Carbonate
1.80	1.78	1.92	Di-calcium phosphate
0.22	0.24	0.27	Salt
0.21	0.24	0.28	DL- Methionine
0.07	0.13	0.12	L-Lysine
0.07	0.08	0.10	L-Threonine
0.50	0.50	0.160	Vitamin-mineral premix1
0.15	0.15	0.15	Sodium bicarbonate
		Analysis results of nutrients
3180	3108	3035	AMEn (kcal/kg)
18	19	21	CP (%)
0.84	0.84	0.9	Ca (%)
0.42	0.42	0.45	Available Phosphorus (%)
0.74	0.80	0.88	Met + Cys
0.95	1.05	1.18	Digestible Lys (%)
0.65	0.69	0.77	Digestible Thr (%)

• ¹
  Premix contain: 4400000 IU/kg of Vit. A, 2000000 IU /kg of Vit. D₃, 30000 IU /kg of Vit. E, 1200 mg/kg of Vit. K (Menadione), 1200 mg/kg of B₁, 3200 mg/kg of B₂, 24000 mg/kg of Nicotinic Acid, 6000 mg/kg of Pantothenic Acid, 1600 mg/kg of B₆, 60 mg/kg of Biotin, 800 mg/kg of Folic Acid, 6 mg/kg of B₁₂; 6400 mg/kg of Copper, 500 mg/kg of Iodine, 16000 mg/kg of Iron, 48000 mg/kg of Manganese, 120 mg/kg of Selenium, 40000 mg/kg of Zinc.

On day 21 of incubation and day 3 of age, one chick per replicate (5 eggs or 5 chicks for each treatment) were randomly selected. Eggs were opened and fetus or chicks were anesthetized with diethyl ether. Immediately, small intestine was removed and flushed with NaCl (150 mmol/l) to remove the contents. Samples of jejunum (2 cm) were taken at the midpoint between the entry of the bile duct and Meckel's diverticulum and immediately stored in liquid nitrogen for messenger RNA (mRNA) extraction. Jejunum was selected as it is the main section of absorption and is the middle of the small intestine.

On days 1 and 3 of age, two chicks per replicate were randomly selected, anesthetized with diethyl ether and jejunum removed and placed in ice and used for microbial assays.

After sectioning and preparation of the samples, RNA samples were extracted according to the method described by Moghaddam et al. (2011). After extraction, RNA purity was determined by calculating the ratio of the absorbance readings at 260 and 280 nm. Additionally, the quality of RNA was assessed by visualization of distinct bands after gel electrophoresis with ethidium bromide staining. A quantity of 1ug of each RNA sample was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcriptase kit (Bioneer Co., Seoul, South Korea). The 20 μL cDNA synthesis reaction contained in addition to the RNA template, 1 μl oligodT (CinnaGen, Iran), 1 μl of 10 mM dNTP (CinnaGen, Iran), 1 μl of Random hexamer (CinnaGen, Iran), 2 μl of 10X MMuLV and 0.5 μl Enzyme M-MLV (Moloney murine leukemia virus). Nuclease-free water was added bringing the reaction up to final volume (20 μl). The mixture was incubated for 1 hour at 42 °C (Moghaddam et al ., 2011). The resulting cDNA was stored at -20 °C prior to use.

Real time PCR was performed for mucin-2 and reference genes at a final volume of 20 μl. This volume contained of 100 ng cDNA, 12.5 μl SYBR^(r) Green Real-Time PCR Master Mixes (Applied Bio System), 1 μl of primers 10 mmol/μl and 4.5 μl distilled water. Amplification of the mucin-2 gene was performed for 40 cycles, which consisted of an initial activations step (95 °C, 5 min), denaturation cycle (95 °C, 15s) and combined annealing and extension (60 °C, 60s). The GAPDH reference gene was amplified at 40 cycles under the same conditions in a different tube. After each run, preparation of standard curve was performed by serial dilution of pooled cDNA from samples. The sequences of the primers used for expression analysis were Mucin chicken F: 5′-CAGCGTTAACACAGGGCTTA-3′, Mucin chicken R: 5′-GCAGCAGACGTTGAT CTCAT-3′, GAPDH Chicken F: 5′-TCTCTGGCAAAGTCCAAGTG-3′, GAP-DH Chicken R: 5′- TGCCCATTGATCACAAG TTT-3′. The relative changes in gene expression were analyzed by using the 2^-ΔΔCt method. Results are expressed as fold-change relative to the control group (Livak & Schmittgen, 2001).

The populations of Escherichia coli and lactic acid bacteria in jejunal contents were estimated as cfu per gram. In sterile condition, sterilized PBS (99 ml) was added (1:100) to 1 g of jejunal content, and then subsequent dilutions prepared. E. coli was cultured on MacConkey agar (Merck, Germany) at 37 °C for 24 hours, and the presence of E. coli then determined. Lactic acid bacteria were enumerated on MRS (Merck, Germany) agar after incubation under anaerobic condition for 72 hours at 37 °C (Witkamp and Olson, 1963).

The statistical normality of all data were tested in MINITAB^(r) software (confidence level=95%). Then treatments analyzed by ANOVA procedure using the GLM procedure of SAS^(r) software. When significant differences among means were found, means were separated using Duncan's Multiple Comparison test (a=5%) for post hoc multiple comparisons.

RESULTS

Hatchability of different treatments was 98.5, 96, 95.5 and 95% for control and three infused groups, respectively. There were no significant differences among treatments for hatchability.

The effects of in ovo infusion of different probiotic strains on the mucin gene expression on day 21 of incubation and day 3 of post-hatch are presented in Table 2. On both days of measurements, a significant difference (p<0.05) was found among treatments. Infusion of Bacillus subtilis induced mucin gene expression 4 and 3.7 folds higher than the control group in day 21 of incubation and day 3 of post-hatch, respectively. Infusion of Pediococcus acidilactici increased the gene expression, but its effect was lower than Bacillus subtilis .

Table 2
Effects of in ovo infusion of different probiotic strains on the expression of mucin gene in the jejunum of pre- and post-hatch chicks

Treatments	Pre-hatch		Post-hatch
	day 21		day 3
Control	1.00 b		1.00 b
Bacillus subtilis	4.16 a		3.76 a
Enterococcus faecium	1.84 b		1.78 b
Pediococcus acidilactici	2.02 ab		2.61 ab
p value	0.002		0.001
SEM	0.343		0.308

• ^a,b
  Means with different superscripts within the same column differ significantly (p<0.05).

Effect of in ovo infusion probiotics on lactic acid bacteria population in the jejunum of broiler chicken is presented in Figure 1A. The population of lactic acid bacteria in the jejunum of one-day old chicks was not affected (p>0.05) by in ovo infusion, but a difference appeared on day 3 of post-hatch (p<0.05). The highest lactic acid bacteria population of in ovo infused groups was for Pediococcus acidilactici and Bacillus subtilis on day 3 of age.

Figure 1
Total lactic acid bacteria (A) and E. coli (B) population in the jejunum of chicks at days 1 and 3 of age

Figure 1B represents the effect of in ovo infusion of probiotics on E. coli population in the jejunum of broiler chicken. Infusion of probiotics had no effect (p>0.05) on E. coli population in one-day old chicks, but decreased its population on day 3 of age, compared with the control group. No differences (p>0.05) were found among different probiotics for E. coli population on day 3 of age.

Effects of in ovo infusion of probiotics strain on daily feed intake, average gain and feed conversion ratio are presented in Table 3. There were no significant differences (p>0.05) among treatments for performance parameters at different periods or in the whole period.

Table 3
Average feed intake (g/day), average weight gain (g/day) and feed conversion ratio of broiler chickens infused probiotic strains

Periods*	d 1-10	d 1-42	d 1-10	d1-42	d 1-10	d 1-42
Control	31.0	109	22.0	54	1.41	2.02
Bacillus subtilis	30.5	110	21.4	55	1.43	2.01
Enterococcus faecium	31.7	106	21.5	53	1.48	1.99
Pediococcus acidilactici	31.2	111	21.8	55	1.44	2.02
p value	0.466	0.328	0.710	0.667	0.268	0.941
SEM	0.497	2.10	0.438	1.21	0.023	0.037

• *d 1-10: starter; d1-42: whole period
• Means without superscripts within the same column not differ significantly (p<0.05).

DISCUSSION

In this study, in ovo infusion of Bacillus subtilis into the amnionic fluid increased the expression of mucin gene on day 21 of incubation and day 3 of post-hatch. The gut mucin amount and composition are arranged based on bacterial glucodisase, activities responsible for removal of monosaccharide residues from mucin carbohydrate side chains (Hoskins et al ., 1985), as well as via regulation of intestinal glycosylation (Bry et al., 1996; Bryk et al., 1999; Freitas et al., 2005; Sharma & Schumacher, 1995). An increased mucin synthesis and secretion have been shown to occur with probiotics consisting mainly of Lactobacillus and Bifidobacterium (Mack, 1999; Mack 2003). According to the study of Tsirtsikos et al. (2012), mucus layer thickness is increased linearly with probiotic inclusion level in the duodenum of chickens on day 14 and 42 of age.

Before hatching, digestive tract of chicks is free of microorganism; early replacement of beneficial bacteria in gut can prepare suitable conditions for cloning of normal microflora and improving quality and health of the gut. Our results indicated that infusion of the probiotic strains into amniotic fluid increased lactic acid bacteria and decreased E. coli populations on day 3 of post-hatch. Lourenco et al. (2012) indicated that oral feeding Bacillus subtilis decreased significantly Salmonella population in broiler gut. Probiotic improves performance and health of bird due to balance of microbial population (Awad et al., 2009). Pediococcus acidilactici prevents growth and development of intestinal small bacteria such as Shigella , clostridium and E. coli . Therefore, Pediococcus acidilactici increases the resistance of birds to pathogenic bacteria (Lee et al., 2007).

In this study, performance parameters were not affected by in ovo infusion. In the numerous studies, the probiotic effects appeared in a three weeks period after addition to the diet (Ghasemi et al., 2010). An infusion in the late fetus period could not affect on performance, and following it, feeding of probiotic is necessary to achieve better results.

As a conclusion, it seems that in ovo infusion of probiotic strains to the late-term fetus has no effect on performance parameters, but has a benefit effect on mucin gene expression and beneficiary microbial colonization in the jejunum of chicks. The best strain, considering mucin gene expression, among the strains used in this study was Bacillus subtilis and for beneficiary bacteria colonization was Bacillus subtilis and Pediococcus acidilactici .

ACKNOWLEDGEMENTS

The authors are grateful to the Islamic Azad University for research funding support. We also thank all the staffs in the Poultry unit, for the assistance in the care and feeding of chicks used in this research.

REFERENCES

• Applegate TJ, Klose V, Steiner T, Ganner A, Schatzmayr G. Probiotics and phytogenics for Poultry:Myth or reality? Journal of Applied Poultry Research 2010;19:194-210.
• Awad WA, Ghareeb K, Abdel-Raheem S, Böhm J. Effects of dietary inclusion of probiotic and synbiotic on growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poultry Science 2009;88:49-56.
• Bry L, Falk PG, Midtvedt T, Gordon JI. A model of host-microbial interactions in an open mammalian ecosystem. Science 1996;273:1380-1383.
• Bryk SG, Sgambati E, Gheri G. Lectin histochemistry of goblet cell sugar residues in the gut of the chick embryo and of the newborn. Tissue Cell 1999;31:170-175.
• Crawford JS. Probiotics in animal nutrition. Proceedings of the Arkansas Nutrition Conference; 1979; Arkansas. USA. p.45-55.
• Cukrowska B, Lodinova-Zadnikova R, Enders C, Sonnenborn U, Schulze J, Tlaskalova-Hogenova H. Specific proliferative and antibody responses of premature infants to intestinal colonization with nonpathogenic probiotic E. coli strain Nissle 1917. Scandinavian Journal of Immunology 2002;55:204-209.
• Cobb. Broiler performance and nutrient supplement guide. Siloam: Cobb-Vantress; 2012. Available from: http://cobb-vantress.com/academy/product-guides.
• Dharmani P, Srivastava V, Kissoon-Singh V, Chadee K. Role of intestinal mucins in innate host defense mechanisms against pathogens. Journal of Innate Immunity 2009;1:123-135.
• Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM. Characterization of Bacillus probiotics available for human use. Applied Environment Microbiology 2004;70:2161-2171.
• Forstner JF, Forstner GG. Gastrointestinal mucus. In: Johnson LR, editor. Physiology of the gastrointestinal tract. New York: Raven Press; 1994. p.1255-1284.
• Freitas M, Axelsson LG, Cayuela C, Midtvedt T, Trugnan G. Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo. Histochemical Cell Biology 2005;124:423-433.
• Fuller R. A review: probiotics in man and animals. Journal of Applied Bacteriology 1989;66:365-378.
• Ghasemi HA, Shivazad M, Esmaeilnia K, Kohram H, Karimi MA. The effects of a synbiotic containing Enterococcus faecium and inulin on growth performance and resistance to coccidiosis in broiler chickens. Journal of Poultry Science 2010;47:149-155.
• Hashemzadeh Z, Karimi-Torshizi MA, Rahimi S, Razban V, Zahraei-Salehi T. Prevention of Salmonella colonization in neonatal broiler chicks by using different routes of probiotic administration in hatchery evaluated by culture and PCR techniques. Journal of Agriculture Science and Technology 2010;12:425-432.
• Hoskins LC, Agustines M, McKee WB, Boulding ET, Kriaris M, Niedermeyer G. Mucin degradation in human colon ecosystems. Isolation and properties of fecal strains that degrade ABH blood group antigens and oligosaccharides from mucin glycoproteins. Journal of Clinical Investment 2010;75:944-953.
• Lee SH, Lillehoj HS, Dalloul RA, Park DW, Hong YH, Lin JJ. Influence of Pediococcus-based probiotic on coccidiosis in broiler chickens. Poultry Science 2007;86:63-66.
• Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and 2-??ct method. Methods 2001;25:402-408.
• Lourenco MC, Kuritza LN, Westphal P, Muniz E, Pickler L, Santin E. Effects of Bacillus subtilis in the Dynamics of Infiltration of Immunological Cells in the Intestinal Mucosa of Chickens Challenged with Salmonella Minnesota. International Journal of Poultry Science 2012;11:630-634.
• Mack DR, Michail S, Wie S, McDougall L, Hollingsworth MA. Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression. American Journal of Physiology 1999;276:G941-950 .
• Mack DR, Ahrne S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion follows adherence of Lactobaccilus strains to intestinal epithelial cells in vitro. Gut 2003;52:827-833.
• Moghaddam HS, Moghaddam HN, Kermanshahi H, Mosavi AH, Raji A. The effect of threonine on mucin2 gene expression, intestinal histology and performance of broiler chicken. Italian Journal of Animal Science 2011;10:66-71.
• Mountzouris KC, Tsitrsikos P, Palamidi I, Arvaniti A, Mohnl M, Schatzmayr G, et al. Effects of probiotic inclusion levels in broiler nutrition on growth performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora composition. Poultry Science 2010;9:58-67.
• Mountzouris, KC, Tsirtsikos P, Kalamara E, Nitsch S, Schatzmayr G, Fegeros K. Evaluation of the efficacy of a probiotic containing Lactobacillus, Bifidobacterium, Enterococcus, and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities. Poultry Science 2007;86:309-317.
• Park JH, Kim IH. Supplemental effect of probiotic Bacillus subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and breast meat quality of growing broiler chicks. Poultry Science 2014;93(8):2054-2059.
• Sharma R, Schumacher U. Morphometric analysis of intestinal mucins under different dietary conditions and gut flora in rats. Digestive Disease Science 1995;40:2532-2539.
• Sterzo E, Paiva J, Penha-Filho R, Berchieri-Junior A. Time required to protect the intestinal tract of chicks against Salmonella enterica serovar Enteritidis using competitive exclusion. Brazilian Journal of Poultry Science 2005;7:119-122.
• Tako E, Ferket PR, Uni Z. Effects of in ovo feeding of carbohydrates and beta-hydroxy-beta-methylbutyrate on the development of chicken intestine. Poultry Science 2004;83:2023-2028.
• Tsirtsikos P, Fegeros K, Balaskas C, Kominakis A, Mountzouris KC. Dietary probiotic inclusion level modulates intestinal mucin composition and mucosal morphology in broilers. Poultry Science 2012;91:1860-1868.
• Uni Z, Ferket PR. Methods for early nutrition and their potential. World's Poultry Science Journal 2004;60:101-111.
• Uni Z, Smirnov A, Sklan D. Pre- and posthatch development of goblet cells in the broiler small intestine:Effect of delayed access to feed. Poultry Science 2003;82:320-327.
• Weis J, Hrncar C, Pal G, Baranska B, Bujko J, Malikova L. Effect of probiotic strain Enterococcus faecium M74 supplementation on the carcass parameters of different hybrid combination chickens. Scientific Papers on Animal Science and Biotechnology 2011;44:149-152.
• Witkamp M, Olson JS. Breakdown of confined and non-confined oak litter. Oikos 1963;14:138-147.
• Zulkifli I, Abdullah N, Azrin M, Ho YW. Growth performance and immune response of two commercial broiler strains fed diets containing Lactobacillus cultures and oxytetracycline under heat stress conditions. British Poultry Science 2000;41:593-597.

Publication Dates

• Publication in this collection
  Jan-Mar 2017

History

• Received
  Apr 2016
• Accepted
  Oct 2016