Effects of Escherichia coli Nissle 1917 on immunity, blood constituents, antioxidant capacity, egg quality and performance in laying Japanese quail

Abstract

This study was conducted to evaluate the effects of E.coli Nissle 1917 (EcN) on immune responses, blood parameters, oxidative stress, egg quality, and performance of laying Japanese quail. A total of one-hundred day-old quail chicks were assigned to 1 of 4 treatments based on probiotic concentration: 1 (0 CFU/mL; control), 2 (10⁴ CFU/mL), 3 (10⁶ CFU/mL), and 4 (10⁸ CFU/mL). The average egg production throughout the 8 wks of the laying phase increased with the increase in EcN supplementation (R²= 0.96). The egg production was also significantly different in wk 4, 7, and 8 of the laying phase with the control group demonstrating the poorest performance. External egg quality parameters, including shell strength, Roche index, albumen height, yolk weight, shell weight, shell thickness, and Haugh unit, also showed higher values in groups receiving EcN. Likewise, these groups demonstrated heavier egg weight (EW) and lower MDA in the meat sample. The MDA concentration of fresh yolk samples in groups receiving EcN was higher but diminished after iron-inducing (P < 0.05). No differences were observed in ADFI, FCR, EM, yolk cholesterol and triglyceride, hematocrit, SRBC, NDV, CMI immune responses (DNCB and PHA), H:L ratio, and internal organ weights. Plasma cholesterol and uric acid were the only blood constituents showing higher values in male birds in groups of EcN (P < 0.05); no appreciable differences were observed in the rest of the blood parameters in male birds and none in females. Further, a substantially higher Avian Influenza Virus (AIV) titer resulted from treatments 3 and 4 in male birds among humoral immune responses. These results demonstrated that EcN in the birds’ drinking water could profoundly influence laying performance, egg quality, immune function, and enhanced antioxidant capacity.

Introduction

Bacterial cultures have always been a proper choice in poultry to either fight pathogens or use as an alternative to antibiotics, and they have shown profound effects on poultry health (Huff et al., 2006). One of the prime candidates in such cultures is probiotics, which are renowned for their antagonistic activity against intestinal pathogens by producing organic acids and bacteriocins (Altenhoefer et al., 2004). Most microorganisms that are commercially dubbed ‘probiotics’ are Gram-positive species; however, a few Gram negatives are also considered probiotics, among which Escherichia coli Nissle 1917 (EcN) is a prime example and a well-defined probiotic for almost a century. It is widely used in a variety of human intestinal diseases, such as chronic constipation and colitis treatment (Huff et al., 2006), and is commercially available as Mutaflor (Ardeypharm, Germany) (Hlinková et al., 2016). EcN is demonstrated to be a stimulus to innate immunity via upregulating the beta-defensins mechanisms and acts as a non-pathogenic bacterium (Huff et al., 2006), which in turn affects the bacterial invasion of epithelial cells (Altenhoefer et al., 2004), improving host defense against bacterial and fungal threats, and eventually improving the clinical signs of ulcerative colitis and Crohn's disease (Huff et al., 2006).

The beneficial effects of EcN on the gastrointestinal tract (GIT) development, intestinal function (Geervliet et al., 2022; Sha et al., 2014), and immune response in humans (Altenhoefer et al., 2004) and pigs (Deng et al., 2014; Geervliet et al., 2022) have already been well documented. Furthermore, some researchers have found EcN intervention to Salmonella Typhimurium invasion (Altenhoefer et al., 2004) and its effect to hinder the invasion of enterotoxigenic Escherichia coli (ETEC) into the jejunal epithelial cells when it administrated orally in piglets (Deng et al., 2014).

GIT maturation enhancement in young turkey poults has been reported by means of EcN (Moyle et al., 2012). EcN has also been demonstrated to enhance innate and adaptive immune responses (Michael et al., 2021), show prophylaxis effects, and treat neonatal diarrhea in calves (Von Buenau et al., 2005), as well as result in reduction of Salmonella Enteritidis in chicken (Huff et al., 2006). It has beneficial effects on intestinal inflammation and influences the human immune system (Sturm et al., 2005). In pigs, EcN revealed a strong influence over reducing the invasion of Salmonella Typhimurium and the adhesion of porcine EPEC with epithelial cells in vitro (Kleta et al., 2006). Using polarized intestinal T84 human epithelial cells as a model system for the intestinal barrier, some have found that EcN provokes an overriding signaling impact which leads to restoration of a disrupted epithelial barrier (Zyrek et al., 2007). Therefore, despite ample investigation into administration of EcN in human studies including, but not limited to, reducing proinflammatory cytokines and treatment of hepatic encephalopathy (Manzhalii et al., 2022), hyperuricemia (Zhao et al., 2022), and characterization of the host–microbial communication (Dokladny et al., 2021), comparatively little work, if any, has been performed in birds. Additionally, there is no report pertaining to the performance of laying birds, egg quality, or blood biochemical analysis. Thus, the objective of current study was to examine these characteristics as well as oxidative status in laying Japanese quails. We hypothesized that EcN could improve laying performance and oxidative status, pave the path to practical use of EcN in poultry industry.

Materials and methods

Birds, diets, and experimental procedure

The Animal Care and Use Committee of Tarbiat Modares University, Tehran, Iran, approved all procedures relating to the use of live birds in this study (approval #: 2538/82). A total of 100 1-day-old unsexed Japanese quail chicks (8.03 ± 0.33 g) were assigned randomly into 4 experimental groups with 5 replicate cages of 5 birds each in a randomized complete block design (RCBD). Treatments were included the control group (1; no EcN), 10⁴ (2), 10⁶ (3), and 10⁸ CFU/mL (4) of EcN added to drinking water daily for 14 wks. Birds were provided ad libitum access to feed and water. Treatments were applied to 1-day-old chicks, and sampling for the laying phase was performed from 7 to 14 weeks of age. Each cage (wire floor: 45 × 40 × 30 cm) was furnished with an electrical bulb on incessant lighting and supplemental heat controlled by an electrical dimmer. The temperature of 35°C was provided on arrival of chicks for the initial 3 d with a subsequent gradual decrease of 2.5°C per week until a constant temperature of 22.5°C was achieved in the rearing phase. After being sent to the production house, the birds were kept under a lighting schedule of 17L: 7D throughout the laying phase, with a male: female at 1:4 in each cage. The basal diet was formulated to meet NRC Nutrient Requirements of Poultry (National Research Council, 1994) specifications for Japanese quail (Table 1).

Table 1.

Ingredients and nutrient composition of starter, grower, and breeder diets (as-fed basis).

Item	Starter and Grower (0 to 42 d)	Breeder (43 to 105 d)
Ingredient, %
Yellow corn	55.57	57.46
Soybean meal (44 % CP)	40.86	33.63
Soybean oil	0.00	1.44
CaCO3 (38 %)	1.26	5.44
Dicalcium phosphate	0.72	1.08
Corn gluten meal	0.51	0.00
Sodium chloride	0.33	0.33
Vitamin premix1	0.25	0.25
Mineral premix2	0.25	0.25
DL-methionine	0.12	0.12
Threonine	0.11	0.00
Lysine	0.02	0.00
Calculated composition3
ME, kcal/kg	2,800	2,800
CP, %	23.17	19.31
Calcium, %	0.77	2.41
Nonphytate phosphorus, %	0.29	0.34
Met, %	0.48	0.43
Met + Cys, %	0.85	0.76
Lysine, %	1.26	1.05
Threonine, %	0.98	0.74
Sodium, %	0.14	0.14

¹The vitamin premix supplied the following per kilogram of complete feed: vitamin A, 9,000 IU (retinyl acetate); D₃, 2,000 IU; vitamin E, 12.5 mg (dl-a-tocopheryl acetate); vitamin B12, 0.015 mg; menadione, 1.76 mg; calcium d-pantothenate, 6.4 mg; biotin, 0.12 mg; pyridoxine, 1.97 mg; thiamine, 1.2 mg; nicotinic acid, 28 mg; riboflavin, 3.2 mg; choline chloride, 320 mg; cyanocobalamine, 0.01 mg; folic acid, 0.38 mg;.

²The mineral premix supplied the following per kilogram of complete feed: manganese (MnSO4·H2O), 60 mg; zinc (ZnO), 51.74 mg; iron (FeSO4·7H2O), 80 mg; copper (CuSO4·5H2O), 8 mg; selenium (Na2SeO3), 0.2 mg; iodine (Iodized NaCl), 0.8 mg.

³Calculated from NRC (1994).

Media preparation

EcN (DSM 6601, serotype O6:K5:H1) was supplied by Ardeypharm GmbH (Herdecke; Nordrhein-Westfalen) which was cultured in tryptic soy broth (Culture medium per liter included 17.0 g tryptone (pancreatic digest of casein), 3.0 g soytone (peptic digest of soybean meal), 2.5 g glucose (dextrose), 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 1000 ml purified water and adjusted pH to 7.3 ± 0.2) for 12 h in a shaking incubator (37 °C, 240 rpm) per manufacturer's recommendation. The culture was then centrifuged for 2 min at 4 °C (10,000 rpm), and the cells were collected, resuspended in sterile water, and diluted to reach 10⁴ CFU/mL. Various concentrations of experimental groups were then prepared and offered on a daily basis.

Egg-related sampling procedures

Egg records of each experimental unit, such as counting and weighing, were documented daily to calculate egg production (EP, %) and egg weight (EW, g/egg). Feed intake was also recorded weekly to measure the average daily feed intake (ADFI, g/hen/day). Next, the egg mass (EM, g/hen/day; EW × EP) was calculated through which the feed conversion ratio (FCR, g feed/g egg laid; ADFI/EM) was determined. Reported values are on a per quail basis.

Ten eggs per group (2 eggs/cage) were collected for physical quality assessment of the eggs. The eggs were weighed intact, and the shell thickness was measured in mm, being the average of the small end, the large end, and the middle of the egg through an ultrasonic device (Echometer, Model 1061; Robotmation). Then, in order to measure the shell-breaking strength (kgf), an eggshell force gauge (Model-II, Robotmation, Tokyo, Japan) was used. After breaking, albumen height (mm), Haugh unit (HU), and the yolk color were determined using an egg multi-tester (EMT-5200; Robotmation). The following formula was used to determine the HU:

$$\mathit{HU}=100\mathit{Lo}{g}_{10}(h+7.57-1.7W^{0.37})$$

where h is albumen height (mm), and W is egg weight (g).

The shells were carefully rinsed and dried for 72 hr at room temperature before weighing by a digital scale (0.001 g). Egg yolk weight was also recorded. A comparison of the yolk color to the Roche yolk color fan was performed to measure the yolk color. The same person made all the egg quality measurements during the experiment to avoid any subjective influence.

The birds were randomly selected (5 birds per treatment) at the end of the experiment and subjected to 4-h preslaughter fasting to evaluate carcass yield and the internal organ weights. The birds were weighed and slaughtered through excising jugular veins. After 2-min bleeding and dipping in a hot water bath for 60 s at 60°C, the birds were de-feathered manually. Upon removing the head and feet, the carcasses were eviscerated, and their weights were recorded. The weight of carcass (without giblet), abdominal fat, proventriculus, gizzard, liver, spleen, testis, heart, and the lengths of duodenum, jejunum, and ileum were recorded and expressed as a percentage of live body weight. The same individual carried out all dissections to minimize variations.

Blood sampling and biochemistry analyses

Prior to slaughtering, the blood samples were collected from the heart using sterilized and heparinized syringes. Following the measurement of packed cell volume via microhematocrit capillary tube, the plasma specimens were harvested by centrifugation (1000 × g for 20 min) and frozen at −20 °C for further analysis (Sedaghat et al., 2016). Total protein, glucose, calcium, albumin, triglyceride, cholesterol, uric acid, and phosphorus were determined spectrophotometrically by using commercially available kits (Pars Azmun, Tehran, Iran) and a microplate reader (Awareness Technology Inc., State Fax 3200, Palm City, FL, USA).

Cell-Mediated Immunity Assay

The cellular immune response was assessed by cutaneous basophilic hypersensitivity (CBH) test using 2,4-Dinitro 1-chlorobenzene (DNCB 10 mg/ mL; vehicle: acetone/olive oil (4:1 v/v)) (Merck; Darmstadt, Germany) and phytohemagglutinin-P (PHA-P, lectin from Phaseolus vulgaris) according to Sedaghat and Karimi Torshizi (2017). At the end of the experiment, the birds were inoculated intradermally with 0.04 mL of the mitogen PHA-P into the right wing-web, and the left wing-web was considered the sham control injected solely with sterile phosphate-buffered saline. The CBH response to DNCB was measured in a ∼1 cm² featherless area on the lateral abdomen by anointing with 0.1 mL of DNCB on the right side, with the left side being treated with the vehicle serving as control. The reaction to PHA-P and DNCB was assessed by measuring skin thickness via a digital micrometer to the nearest 0.001 mm immediately before (0 h) and 24 h after injection. The formula with which the swelling induced in the area as the obtained response to mitogen was calculated is as follows:

$$\frac{\left(RPRE-RPOST\right)-\left(LPRE-LPOST\right)}{IT}$$

Where RPRE is the right side pre-injected thickness, RPOST is the right side post-injected thickness, LPRE is the left side pre-injected thickness, LPOST is the post-injected thickness, and IT is the initial thickness.

Humoral immune response

Upon collecting the whole blood, smear samples on glass slides were prepared using one drop of blood to determine the heterophil-to-lymphocyte (H:L) ratio. The specimens were then air-dried and stained with Wright's-Giemsa (Saikin Kagaku Institute Co. Ltd, Sendai, Japan). A total of 100 white blood cells per specimen, including heterophil and lymphocyte cells, were enumerated differentially with a light microscope (standard 20; Carl Zeiss, Göttingen, Germany) at 1000 × magnification. The H:L ratio was then calculated by dividing the number of heterophils obtained by the number of lymphocytes.

Newcastle disease virus vaccine (NDV; Live B1 strain; Vetrina, Zagreb, Croatia) and avian influenza virus (AIV) were performed, respectively, via an eye dropper and through subcutaneous injection as described by the manufacturer's recommendations. In order to detect the antibodies against NDV and AIV in the plasma of immunized quails at the end of the experiment, the antibodies commercial test kits were used by haemagglutination inhibition (HI) assay. The assay was carried out as described by the manufacturer's instructions on the collected plasmas. First, 8 haemagglutination (HA) units of AIV and 4 HA units of NDV were added to a microtiter (25 µl/well). The highest dilution of the virus was determined as 1 HA unit, which agglutinates chicken red blood cell (RBC; 1:100) suspension at 25°C under the pH of 7.2. Plasma (25 µL) was then added to the microtiter comprising 25 µL PBS in order to achieve 2-fold serial dilutions. Later, 25 µL of chicken RBC suspension (1 %) per well was included and set at 37°C for 45 min. Finally, log₂ of the reciprocal of the last plasma dilution inhibiting the HA activity was expressed as the HI titer.

A suspension of sheep red blood cells (SRBC; 5 % v/v PBS) as the T-cell-dependent antigen was prepared and injected intramuscularly (0.1 mL/bird) with a booster injection 7 d later to determine the secondary anti-SRBC antibody response. After collecting the whole heparinized blood samples 7 days following the booster, the assessment of anti-SRBC antibody titer was performed by the microhaemagglutination test via a 96-well microtiter plate. The plasma specimens were first inactivated at 56°C for 30 min. Then, 25 µL of both plasma and PBS were transferred in the first row of the microtiter plate and incubated at 37°C for 30 min, and the rest of the wells were supplied with 25 µL of PBS to serve as a 2-fold serial dilution of each specimen on consecutive rows. Next, the microtiter plate was reincubated at 37°C for 30 min after adding 25 µL of an SRBC suspension to each well. The plate was then monitored by the haemagglutination test. The log₂ of the reciprocal of the highest dilution, giving a detectible agglutination, was reported as obtained antibody titers.

Determination of TBARS value in yolk and meat sample

At the end of the experiment, eggs from each experimental unit were collected, and the yolk samples were stored following the egg quality assessments. Individual carcasses from 2 birds per cage (10 birds per treatment) were trimmed for thigh muscle specimens. A spectrophotometric approach was performed to determine the malondialdehyde (MDA) of specimens (Sedaghat et al., 2016). A thoroughly homogenized mixture of the specimen, 5 mL of butylated hydroxytoluene in hexane (8 g/L) and 8 mL of trichloroacetic acid (50 g/L) was prepared before centrifuging for 3 min at 3000 × g. The achieved supernatant fluid, hexane, was discarded. Then, 1.5 mL of aqueous 2-thiobarbituric acid (8 g/L) was mixed with a 2.5 mL aliquot from the bottom layer and incubated for 30 min at 70°C. After incubation, the blend was cooled to room temperature and submitted to the conventional spectrophotometry at 532 nm.

The iron-induced lipid oxidation was also performed to evaluate the extent to which the lipid oxidation of specimens could develop in a commercially stored setting. Briefly, a stock solution comprising 1.138 mM ferrous sulfate and 0.368 mM ascorbic acid was prepared and added to each specimen (1.5 mL), followed by incubation for 60 min at 37°C. Next, the blend of iron-induced specimens and non-induced specimens was let to cool off to room temperature and submitted to MDA assay via conventional spectrophotometry in the 532 nm.

Statistical analysis

Data were subjected to ANOVA using the GLM procedure of SAS 9.4 (SAS Institute, 2014) according to an RCBD. The model included EcN treatment and block as independent variables. Orthogonal polynomial contrast was conducted to determine the linear and quadratic effects of increasing levels of EcN inclusion. When the model was significant, the Duncan multiple range test was carried out to compare treatment means. The individual cage was considered the experimental unit, and statistical significance was determined at α level of 0.05. The effects are expressed as mean values with pooled SEM. The model for RCBD is represented as:

$$Y_{\mathit{ij}}=\mu +{\tau }_i+{\beta }_j+{\epsilon }_{\mathit{ij}}$$

Where Y_ij is the observed response for treatment "i", EcN levels, in block "j", replicates; µ is the overall population mean; τ_i is the effect of EcN levels; and β_j is the effect of replicates.

ε_ij: is the random error associated with observation "ij"

Results

Egg quality and laying performance

The effect of EcN in drinking water on external egg quality is presented in Table 2. A linearly significant effect was observed in shell strength, in which the shell robustness increased as the concentration of EcN in drinking water increased (P = 0.003). The same linear trend was observed in the Roche index (P < 0.0001), albumen height (P = 0.002), yolk weight (P = 0.002), and shell weight (P = 0.0007). Additionally, supplementing EcN in group 4 yielded a superior shell thickness (0.22 mm; P < 0.05) and Haugh unit (92.34; P < 0.05).

Table 2.

Effect of supplementing drinking water with EcN on the external egg quality parameters in laying Japanese quail ¹.

Item	Shell strength (kgf)	Roche index	Albumen height (mm)	Yolk weight (g)	Shell weight (g)	Shell thickness (mm)	Haugh unit
EcN treatment
1	0.99 b	6.32 b	4.52 b	3.36 b	0.96 b	0.21 b	90.07 b
2	1.10 ab	6.48 b	4.54 b	3.43 b	0.99 ab	0.21 b	90.05 b
3	1.16 a	7.41 a	4.57 b	3.55 ab	1.04 a	0.21 b	89.98 b
4	1.20 a	7.67 a	5.03 a	3.64 a	1.05 a	0.22 a	92.34 a
SEM	0.02	0.09	0.05	0.03	0.009	0.10	0.31
P-value	0.03	<0.0001	0.003	0.02	0.005	0.03	0.01
Orthogonal polynomial contrast
Linear	0.003	<0.0001	0.002	0.002	0.0007	0.02	0.01
Quadratic	0.57	0.76	0.05	0.86	0.55	0.05	0.05

¹Treatments 1, 2, 3, and 4 stand for control (no probiotic), 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

^abMeans with different letters within the same column differ significantly (P ≤ 0.05).

The results of laying performance for 8 weeks of laying phase and the overall average egg production are summarized in Fig. 1, Fig. 2, respectively. Fig. 1 presents an appreciable difference among experimental groups in weeks 4, 7, and 8 of the laying phase, in all of which the control group demonstrated an inferior production rate when compared with EcN-treated groups (P < 0.05).

Fig. 1.

The effects of EcN on the egg production rate of laying Japanese quails during the 8-week production period. Treatments 1, 2, 3, and 4 stand for control (no probiotic), 10⁴, 10⁶, and 10⁸ CFU/mL supplemented probiotic E.coli Nissle 1917 to drinking water.

Fig. 2.

The effects of EcN on overall egg production average of laying Japanese quails during the 8-week production period. Treatments 1, 2, 3, and 4 stand for control (no probiotic), 10⁴, 10⁶, and 10⁸ CFU/mL supplemented probiotic E.coli Nissle 1917 to drinking water.

The overall average egg production rate during the experiment is summarized in Fig. 2. A linear correlation was observed by supplementing drinking water with EcN, in which the average egg production rate increased as the concentration of EcN increased (R²= 0.96). Treatments 1, 2, 3, and 4, respectively, yielded 75.54, 82.33, 83.48, and 85.12 percent average egg production during the 8 weeks of laying performance. The R² for the model of performance as a function of the EcN, along with the equation, is provided in Fig. 2.

ADFI, FCR, EW, EM and EP

The data in Table 3 represent the effects of supplementing the drinking water with EcN on ADFI, FCR, EW, and EM in laying Japanese quails. No appreciable differences were observed in ADFI, FCR, and EM (P > 0.05). However, the EW of the groups that received EcN was heavier than that of the control group (P < 0.05).

Table 3.

Effect of supplementing drinking water with EcN on ADFI, FCR, EW, and EM in laying Japanese quail¹.

Item	ADFI (g/day)	FCR (g/g)	EW (g/egg)	EM (g/bird/day)
EcN treatment
1	36.33	4.34	10.60 b	8.66
2	38.36	3.85	11.54 a	10.51
3	37.00	4.02	11.24 ab	9.37
4	36.06	4.48	11.54 a	8.83
SEM	0.85	0.16	0.13	0.32
P-value	0.81	0.54	0.02	0.17
Orthogonal polynomial contrast
Linear	0.79	0.69	0.01	0.81
Quadratic	0.42	0.17	0.15	0.06

¹EcN, E. coli Nissle 1917; ADFI, average daily feed intake; FCR, feed conversion ratio; EW, egg weight; EM, egg mass. Treatments 1, 2, 3, and 4 stand for control (no probiotic), 10⁴, 10⁶, and 10⁸ CFU/mL supplemented probiotic E.coli Nissle 1917 to drinking water.

^abMeans with different letters within the same column differ significantly (P ≤ 0.05).

Yolk and meat MDA, yolk cholesterol and triglyceride

The effects of EcN on MDA concentration of fresh yolk and meat samples of Japanese quail, yolk cholesterol, and triglyceride are shown in Table 4. The MDA content of fresh meat was substantially decreased by EcN treatments (0.072, 0.036, 0.035, and 0.030 μg/g for treatments 1, 2, 3, and 4, respectively; P < 0.0001). On the other hand, the content of yolk MDA in fresh samples increased by EcN, and the lowest amount belonged to the control group (0.029 μg/g; P = 0.003). However, this value diminished as iron-mediated oxidative stress was induced in yolk samples in groups 3 and 4, demonstrating lower concentrations of MDA (0.126 and 0.149 μg/g, respectively; P = 0.02) than in groups 1 (control) and 2 (0.182 and 0.181 μg/g, respectively). There were no significant differences in yolk cholesterol and triglyceride among experimental groups (P > 0.05), irrespective of numerical decreases in EcN-treated groups.

Table 4.

Effects of supplementing drinking water with EcN on meat and yolk MDA, yolk cholesterol and triglyceride of Japanese quail ¹.

Item	Meat MDA (μg/g)	Y-MDA0 (μg/g)	Y-MDA-I (μg/g)	Y-CHL (mg/g)	Y-TG (mg/g)
EcN treatment
1	0.072 a	0.029 b	0.182 a	12.33	20.54
2	0.036 b	0.060 a	0.181 a	11.37	19.58
3	0.035 b	0.049 a	0.126 b	11.11	19.67
4	0.030 b	0.058 a	0.149 ab	11.23	19.35
SEM	0.003	0.003	0.008	0.31	0.25
P-value	<0.0001	0.003	0.020	0.55	0.42
Orthogonal polynomial contrast
Linear	<0.0001	0.005	0.024	0.25	0.16
Quadratic	<0.0001	0.064	0.393	0.42	0.55

¹Y-MDA0, MDA content of freshly yolk sample; Y-MDA-I, MDA content of iron-induced yolk sample; Y-CHL, yolk cholesterol; Y-TG, yolk triglyceride. Treatments 1, 2, 3, and 4 stand for control (no probiotic), 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

^abMeans with different letters within the same column differ significantly (P < 0.05).

Biochemical analysis

Table 5 illustrates the effects of probiotic EcN on plasma biochemical analysis of male and female birds. None of the female groups produced any significant effect in blood biochemistry, while plasma cholesterol and uric acid contents of male birds were observed to be affected by EcN (quadratic effect in cholesterol with P = 0.008), in which the highest levels of cholesterol were seen in groups 2 and 3 (222.55 and 204.55 mg/dL, respectively). This quadratic effect was also observed in the triglyceride level in male birds, in which groups 2 and 3 resulted in the greatest values (P = 0.009; 207.05 and 208.13 mg/dl, respectively). The plasma uric acid of male birds increased linearly (P = 0.02) with increasing EcN administration. There were no significant differences in the values of other blood parameters in male birds (P > 0.05).

Table 5.

Effects of supplementing drinking water with EcN on some blood characteristics of Japanese quail.

Item1	TP (g/dL)	Glu (mg/dL)	Ca (mg/dL)	Alb (g/dL)	TG (mg/dL)	CHL (mg/dL)	UA (mg/dL)	P (mg/dL)
Female
EcN treatment
1	5.70	130.15	11.59	3.65	611.20	222.95	6.07	8.44
2	5.79	127.69	11.44	4.10	577.11	205.18	6.05	8.76
3	5.41	126.61	11.01	3.42	534.15	190.36	5.87	8.17
4	5.47	137.07	11.20	3.79	596.21	198.62	6.17	8.44
SEM	0.09	5.00	0.16	0.12	16.97	7.38	0.15	0.24
P-value	0.50	0.90	0.66	0.26	0.43	0.48	0.93	0.89
Orthogonal polynomial contrast
Linear	0.24	0.68	0.32	0.80	0.57	0.20	0.93	0.81
Quadratic	0.92	0.55	0.62	0.87	0.17	0.39	0.63	0.95
Male
EcN treatment
1	4.69	131.67	8.29	2.65	178.35	191.93 b	5.41 b	4.25
2	4.70	124.00	8.33	2.67	207.05	222.55 a	5.75 ab	4.07
3	5.08	128.40	8.62	2.72	208.13	204.55 ab	6.58 a	4.25
4	4.82	127.27	8.41	2.66	188.97	195.79 b	6.16 ab	4.32
SEM	0.07	3.45	0.04	0.02	4.73	4.04	0.16	0.11
P-value	0.19	0.90	0.05	0.76	0.05	0.02	0.04	0.90
Orthogonal polynomial contrast
Linear	0.21	0.79	0.09	0.66	0.38	0.82	0.02	0.74
Quadratic	0.33	0.66	0.15	0.46	0.009	0.008	0.19	0.61

¹TP, total protein; Glu, glucose; Ca, calcium; Alb, albumin; TG, triglyceride; CHL, cholesterol; UA, uric acid; P, phosphorus. Treatments 1, 2, 3, and 4 stand for control (no probiotic, 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

^abMeans with different letters within the same column differ significantly (P < 0.05).

Hematocrit and organ weight

Table 6, Table 7 show that there were no significant differences in hematocrit, internal organ weights, and the length of duodenum, jejunum, and ileum among experimental groups, neither in male nor female birds (P > 0.05).

Table 6.

Effects of supplementing drinking water with EcN on hematocrit levels (%) of Japanese quail¹.

Item	Male	Female
EcN treatment
1	48.60	40.40
2	47.60	36.60
3	45.00	41.00
4	44.00	39.40
SEM	1.16	1.16
P-value	0.49	0.58
Orthogonal polynomial contrast
Linear	0.14	0.89
Quadratic	1.00	0.65

^a-b Means with different letters within the same column differ significantly (P < 0.05).

¹Treatments 1, 2, 3, and 4 stand for control (no probiotic, 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

Table 7.

Effects of supplementing drinking water with EcN on carcass yield and the internal organ weights of male Japanese quail¹.

Item	Body weight (g)	% of live body weight								Intestinal sections (cm)
		Carcass	Abdominal fat	Proventriculus	Gizzard	Liver	Spleen	Testes	Heart	Duodenum	Jejunum	Ileum
EcN treatment
1	240.97	62.08	2.28	0.27	1.60	1.41	0.02	2.25	0.71	10.90	18.50	18.50
2	224.37	61.67	3.24	0.33	1.83	1.66	0.03	2.34	0.69	10.60	18.70	18.70
3	220.12	62.72	2.42	0.33	2.11	1.57	0.04	2.40	0.76	10.00	18.50	18.50
4	235.44	62.70	2.55	0.31	1.76	1.63	0.04	2.43	0.76	10.90	19.70	19.70
SEM	4.30	2.72	0.61	0.02	0.13	0.15	0.01	0.10	0.04	0.22	0.33	0.33
P-value	0.30	0.31	0.71	0.64	0.24	0.72	0.32	0.39	0.21	0.49	0.55	0.55
Orthogonal polynomial contrast
Linear	0.58	0.79	0.94	0.42	0.26	0.46	0.10	0.32	0.38	0.77	0.27	0.27
Quadratic	0.07	0.06	0.67	0.36	0.17	0.94	0.81	0.16	0.09	0.21	0.47	0.47

¹Treatments 1, 2, 3, and 4 stand for control (no probiotic, 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

Immune response

The effects of supplementing drinking water with EcN on immune response indices in Japanese quails, including anti-SRBC, AIV, and NDV, are presented in Table 8. There were no appreciable differences in SRBC and NDV response of male birds of different groups, while the response to AIV was significantly influenced by EcN (P = 0.001). The male birds of treatments 3 and 4 yielded the highest antibody titer against AIV, demonstrating a linear response (P = 0.0007). Additionally, the immune response in female birds against SRBC was higher in EcN (P = 0.001) groups than that of the control group, and the response against AIV and NDV did not differ significantly (P > 0.05). Furthermore, the H:L ratio, PHA, and DNCB responses were not affected by EcN treatment in either male or female birds (P > 0.05; data not shown for brevity).

Table 8.

Effects of supplementing drinking water with EcN on humoral immune response of Japanese quail¹.

Item	SRBC	AIV	NDV
Male
EcN treatment
1	1.20	4.60 b	6.80
2	0.40	3.60 b	7.00
3	0.20	7.40 a	5.40
4	0.20	7.60 a	7.40
SEM	0.18	0.50	0.41
P-value	0.17	0.001	0.36
Orthogonal polynomial contrast
Linear	0.05	0.0007	0.95
Quadratic	0.26	0.38	0.28
Female
EcN treatment
1	0 c	7.8	3.4
2	0.6 ab	8.2	1.6
3	1 a	6.6	1.8
4	0.2 bc	6.8	0
SEM	0.11	0.51	0.78
P-value	0.001	0.68	0.53
Orthogonal polynomial contrast
Linear	0.17	0.35	0.17
Quadratic	0.0004	0.92	1.00

¹SRBC, sheep red blood cell; AIV, avian influenza virus; NDV: Newcastle disease virus. Treatments 1, 2, 3, and 4 stand for control (no probiotic, 10⁴, 10⁶, and 10⁸ CFU/mL supplemented E.coli Nissle 1917 to drinking water.

^abMeans with different letters within the same column differ significantly (P < 0.05).

Discussion

Feed supplementation of probiotics is more common in poultry than other methods. However, administering probiotics via drinking water has plethora of advantages described extensively by Karimi Torshizi et al. (2010). They concluded that drinking water supplementation is is superior to more conventional in-feed supplementation methods. There are myriad probiotics on the market that have proven to have effects in many field trials in the poultry sector. However, only a few studies demonstrate the effects of EcN in animal science, and even fewer reports are available on supplementing EcN in poultry species. Therefore, this study is pioneered in evaluating the long-term effects of probiotic EcN in poultry and laying birds and on quality measurements of eggs, antioxidant capacity, immune function, and blood constituents. The efficacy of probiotics depends largely on the type, concentration, bacterial strains, management system, basal diet, and environmental stress factors (Forte et al., 2016). Therefore, it is difficult to compare their effectiveness and, hence, every attempt has been made in this paper to discuss the most relevant ones, if any.

Egg quality and productive performance

Different studies have observed different effects of probiotics on laying performance and egg quality, some reporting positive effects and others have observed none. For instance, Siadati et al. (2018) reported a positive effect of feeding four selected native Lactobacillus strains on egg production, egg mass, FCR, Haugh unit, and albumen height of laying Japanese quails and no significant effects on feed intake, egg weight, shell, yolk, and albumen. Higher egg production and increased eggshell thickness were also achieved in a study using commercially available probiotic preparations comprising strains of bacteria and yeasts in quail breeders (Güçlü, 2011). On the other hand, a study comparing the effects of single and multi-strain probiotics in broiler breeders revealed no significant effects on egg production, egg weight, shell weight, and body weight (Aalaei et al., 2018). These discrepancies might be attributed to the difference in environmental conditions, composition of diet, and, above all, probiotic strain. In the current study, we observed a superior Haugh unit, shell thickness, and albumen height with supplementing EcN, congruent with the study in which lactic acid bacteria were used in laying Japanese quails (Lokapirnasari et al., 2019). Haugh unit and albumen height are the essential internal characteristics of egg freshness as part of the egg quality definition (Lokapirnasari et al., 2019). They appeared to be higher with higher level of EcN. In another study, the positive effects of supplementing Bacillus on laying performance, egg weight, shell thickness, and Haugh unit in laying hens have been reported (Mazanko et al., 2018). Such improvements via probiotics are speculated to be due to the abundance of metabolites and lytic enzymes demonstrating DNA-protective and antioxidant properties. They also referred to bacilli-produced proteases, amylases, and cellulases as the main contributors to improved feed digestion, bringing about these positive outcomes. An improved shell thickness by probiotics has been suggested to arise from increased Ca absorption in the intestine (Güçlü, 2011).

In the current study, egg production and laying performance was improved with an increase in EcN supplementation. The increased egg production via probiotics may be due to a superior intestinal absorptive capacity as a result of elongated small and large intestinal lengths, as well as suppressing the pathogenic bacteria and bolstering the growth and activity of beneficial bacteria (Güçlü, 2011).

In a previous study in which the EcN was encapsulated in soy protein of broiler diets, the EcN (10⁹ cfu/g) resulted in higher BWG and feed intake with lower FCR (Settles, 2021) as well as an enhanced survival rate (Huff et al., 2006). It is also demonstrated that orally administered EcN in piglets increased daily feed intake, daily weight gain, feed conversion, and final body weight (Deng et al., 2014).

Yolk and meat MDA, yolk cholesterol and triglyceride

The results of present study have shown that administration of EcN via drinking water could modulate the oxidative status by alleviating MDA content in the meat and iron-induced yolk samples, indicating a protective effect against oxidative agents in case of preserving the yolk samples for longer times than the fresh ones. These results are in line with the study reporting antioxidant activities of EcN when SOD and T-AOC activities were measured as biomarkers of the antioxidant response of jejunal mucosa in piglets (Deng et al., 2014). Furthermore, they revealed an enhanced jejunal development in piglets via EcN. It is well-established that probiotics could demonstrate antioxidant activity by increasing the activity of SOD and the level of serum MDA in broiler chickens (Raza, 2021). Additionally, probiotics such as B. megaterium, B. subtilis, and B. laterosporus have shown to reduce the MDA content in the presence of aflatoxin B1 and mitigate its deleterious effects in Japanese quails (Razmgah et al., 2020). MDA concentration in serum and tissues is one of the main indicators of peroxidation or oxidative damage to lipids. Oxidation of lipids is due to the abundance of free radicals and reactive oxygen species, which is postulated to be the reason behind elevated MDA concentration (Raza, 2021). Therefore, the current study demonstrates that EcN could alleviate oxidative stress by reducing the MDA level in fresh samples of meat and iron-induced samples of yolk in Japanese quails.

Blood biochemistry

It is documented that probiotics in drinking water positively affect serum biochemical characteristics (Karimi Torshizi et al., 2010; Hashemzadeh et al., 2013). Hashemzadeh et al. (2013) studied the effects of EcN as a Gram-negative probiotic. They compared it with Gram-positive probiotics in broilers and found no significant differences in serum total protein, albumin, triglyceride, and calcium, which are in agreement with our study in which no appreciable differences were observed in the aforementioned biochemical analyses in either male or female birds. They also reported a significant effect on serum uric acid, which is in line with the present study in which we observed significantly higher uric acid levels in male birds. There are no inherent sex-based differences in serum uric acid content of Japanese quail (Scholtz et al., 2009), and hyperuricemia is reported to be related to abdominal fat and hypercholesterolemia in quails (Lin et al., 2009), albeit no significant differences were seen in abdominal fat accumulation. Thus, the reason behind the elevated level of uric acid concurrently with the hypercholesteremic effect via EcN in males might be due to its interference in the metabolism of lipids and hormonal changes in male birds. This claim could be further substantiated via higher hatchability and the higher number of spermatocytes in seminiferous tubules of testis in male birds of EcN-treated groups (unpublished data). Probiotics possess hypocholesterolemic and hypolipidemic properties in various studies, and this acknowledges the results of the current study regarding females despite showing no significant difference and revealing only numerical reduction. However, the male birds' results differed, in which a hypercholesteremic effect was observed. This hypercholesterolemia via probiotics might be due to the inhibition of cholesterol biosynthesis associated with hydroxy‑3-methylglutaryl coenzyme A-3, a key enzyme in the synthesis of cholesterol (Ghasemi-Sadabadi et al., 2019).

Immune response

A boost in immune function, which is shown as higher antibody titers against AIV in male birds and SRBC in females as a result of supplementing EcN, was observed in the current study. An enhancement in immune function through EcN was reported in broiler chickens when the birds were challenged with APEC (Huff et al., 2006). Improved jejunal health has also been demonstrated via oral administration of EcN in challenged piglets with E. coli Abbottstown (Deng et al., 2014). The modes of action of EcN have extensively been reviewed and discussed by Schultz (2008). EcN is believed to interact with the immune system, reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines through peripheral blood mononuclear cells. It may also depress the development of newly recruited T cells into the intestinal mucosa, thereby reducing intestinal inflammation. This event, in turn, does not influence activated tissue-bound T cells, which might lead to the elimination of detrimental antigens to maintain immunological homeostasis (Settles, 2021). Moreover, the robust influence of EcN on intestinal immune function has been reported to be due to a specific lipopolysaccharide responsible for its immunogenicity with no major immunotoxicity (Scaldaferri et al., 2016).

Conclusion

In summary, EcN in drinking water enhanced laying performance, egg quality, antioxidant status, and immune response. The ability of EcN as a single, safe, and well-qualified bacterial strain to improve productive performance and modulate the stress response in poultry is a distinct advantage in the evolution of a probiotic product that could be potentially considered as an antibiotic alternative. Moreover, it is of paramount importance to evaluate its effect against foodborne pathogens of particular importance in poultry, as its prophylactic effects have already been demonstrated against Campylobacter jejuni and are being extensively studied in humans. Hence, further research is warranted to look into EcN effects in more poultry species and stress conditions.

Declaration of competing interest

The authors hereby declare that no actual or potential conflicts of interest, including any personal, financial, or other relationships, neither direct nor indirect, with individuals or organizations, exist that might raise the question of bias in the work reported or the conclusions.