Evaluation of the Effect of In Ovo Applied Bifidobacteria and Lactic Acid Bacteria on Enteric Colonization by Hatchery-Associated Opportunistic Pathogens and Early Performance in Broiler Chickens

Abstract

Probiotics have been used to promote pioneer colonization by beneficial bacteria in poultry. The purpose of the present study (four trials) was to determine if an in ovo injection with Bifidobacterium saeculare (B2-2, B3-4) and/or lactic acid bacteria (LAB18, LAB46) at d18 of embryogenesis reduced Enterococcus and Gram-negative bacterial colonization in the gut at hatch. An environmental challenge model was used to simulate microbial contamination in large-scale hatch cabinets in a laboratory setting. In all trials, gut homogenates obtained from chicks at hatch were plated to enumerate relevant bacterial populations. Performance was evaluated in trials two and four. The top treatments in trial one were selected for further testing in trials 2–4. The significance level for all analyses was set at p < 0.05. A meta-analysis of gut bacterial recovery for trials 2–4 revealed that the administration of probiotic treatments increased LAB and/or bifidobacteria at hatch compared to non-treated groups. LAB46 + B2-2 was more effective at reducing Gram-negative bacteria and Enterococcus in the gut compared to other probiotic treatments. All treatments improved d14 BW and d0–14 BWG compared to non-treated groups. These results indicate that exposure to beneficial bacteria during late embryogenesis can prevent colonization by opportunistic pathogens associated with contamination in commercial hatch cabinets and may also improve early performance in broiler chickens.

1. Introduction

Pioneer colonizers are the first microorganisms to colonize a presumably sterile environment [1], such as the gastrointestinal tract. In mammals, the fetus encounters the initial microorganisms as it passes through the vaginal canal [2]. A similar process occurs during oviposition in avian species, where microorganisms present in the hen’s cloaca or feces are deposited onto the eggshell surface [3]. In nature, among avian species, the transfer of the maternal microbiota is further carried out through close physical contact with the hen during the hatching and brooding phases [4]. Behaviors, such as cloacal drinking and coprophagy [5], may also play a role in pioneer colonization for avian species as these behaviors directly expose the chick to the microorganisms present in the environment. However, in commercial poultry operations, chicks and poults have limited exposure to the hen’s microbiota since eggs will be promptly removed after laying. Instead, naïve chicks are initially exposed to apathogenic and pathogenic microorganisms present in the hatchery environment. Since microorganisms present in the environment during hatch serve as the pioneer colonizers of the gastrointestinal tract of chicks [3,4,6,7], it is critical to control the microbial load in a hatchery setting. Furthermore, promoting early exposure to beneficial microorganisms that colonize the gastrointestinal tract has been shown to reduce the colonization and shedding of pathogenic microorganisms [8]. In chickens, early colonization by beneficial pioneer colonizers has been associated with improved development and maturation of the gut [9,10,11,12], promoted establishment and maturation of the gut microbiome [12,13,14], and regulated immunological functions [15,16,17,18]. As a result, there has been an emphasis on introducing beneficial pioneer colonizing bacteria during late embryogenesis or at hatch in commercial hatcheries. In the hatchery, probiotics may be administered using methods such as an in ovo injection [13,19], spraying the eggs pre-hatch [20,21], or spraying or fogging the chicks post-hatch [22]. Oral administration via gavage or drinking water application immediately post-placement at the farm has also been explored [23]. However, application at the hatchery prior to transportation to the farm has benefits since timing is critical to avoid pioneer colonization by microorganisms of environmental origin. Thus, the purpose of the present study was to evaluate the effect of in ovo administration of lactic acid bacteria and Bifidobacterium saeculare on enteric colonization at hatch and early performance in broiler chickens using an adapted multi-species challenge model [24] to simulate microbial contamination in commercial hatch cabinets in a laboratory setting. We hypothesized that the in ovo application of the probiotic treatments described herein would promote colonization by beneficial bacteria and improve performance in broiler chickens compared to non-treated controls.

2. Materials and Methods

2.1. Experimental Design

Four trials were conducted to evaluate the effect of Pediococcus acidilactici (LAB18), Lactobacillus salivarius (LAB46), and/or two Bifidobacterium saeculare (B2-2, B3-4) administered during late embryogenesis on hatchability, enteric colonization at hatch, and early performance in broiler chicks (Supplementary Table S1). To identify top-performing treatments that reduced enteric colonization by Gram-negative bacteria and Enterococcus spp., individual LAB and bifidobacteria were administered alone or in combination by in ovo injection into the amniotic cavity at 18 days of embryogenesis (DOE18). For trial 1, treatment groups included (1) Non-challenged, Non-treated Control (NC), (2) Pathogen Mix Challenged Control (PM), (3) LAB18, (4) LAB 46, (5) B2-2, (6) B3-4, (7) LAB18 + B2-2, (8) LAB46 + B3-4, (9) LAB18 + B3-4, and (10) LAB46 + B2-2. Treatment groups for trials 2–4 were selected based on trial 1 results. For trials 2–4, treatment groups included (1) NC, (2) PM, (3) LAB46, (4) LAB46 + B2-2, (5) LAB46 + B3-4, and (6) LAB46 + B-Combo.

One hatcher (GQF 1550) was utilized per treatment (n = 240 embryos/hatcher) in trials 1 and 2. For trial 3, two hatchers were used per treatment (n = 180 embryos/hatcher). In trial 4, two hatchers were used for NC and PM, whereas only one hatcher was used for the remaining treatment groups (n = 225 embryos/hatcher). In all experiments, in ovo treatments were administered on DOE18, and challenge was administered to the eggshell at DOE19, as described in detail below. At hatch, hatchability was recorded (trials 2–4), and gut samples were collected to evaluate the enteric colonization of relevant bacteria for all trials. Performance was only evaluated in trials 2 and 4 (trial 2 n = 8 pens/treatment, n = 20 chicks/pen; trial 4 n = 6–12 pens/treatment, n = 22 chicks/pen). The allocation process was performed to normalize and prevent treatment effects on body weight (BW) by providing similar (within 10 g) starting pen weights. Chicks were placed in replicate 2 × 2 ft floor pens by treatment on fresh pine shavings. To minimize cross-contamination, gloves were changed between treatments, and all surfaces being used (scale, tubs for weighing, etc.) were disinfected with 70% ethanol. Pen BW was recorded on the day-of-hatch (DOH), d7, and d14 to evaluate average body weight (BW) and body weight gain (BWG). Feed was weighed at d0, d7, and d14 to calculate the feed conversion ratio (FCR). Chicks were provided ad libitum access to water and a balanced, unmedicated corn and soybean meal diet meeting nutritional requirements for broilers recommended by Aviagen [25].

2.2. Lactic Acid Bacteria

LAB (LAB18 and LAB46), derived from Flora-Max B11 (Pacific Vet Group, Selangor, 47500 Petaling Jaya, Malaysia), were used in these trials and have been shown to reduce Salmonella colonization and improve performance in broiler chickens when administered by an in ovo injection into the amniotic cavity at DOE18 [19]. LAB18 and LAB46 have been identified as Pediococcus acidilactici and Lactobacillus salivarius, respectively [19]. To prepare each LAB isolate for in ovo injections, 100 mL of Man Rogosa Sharpe (MRS Agar, Hardy Diagnostics, Santa Maria, CA, USA, Cat. No. C5932) broth was inoculated with a 1 mL aliquot and incubated aerobically at 37 °C for 24 h. After incubation, a subsample of the turbid culture was removed to enumerate the stock concentration (CFU/mL). Stock concentration was determined by drop plating onto MRS agar followed by incubation at 37 °C for 24 h. The remaining stock was stored at 4 C overnight. A final LAB concentration for in ovo treatments of 1 × 10⁴ CFU/embryo was confirmed by drop plate enumeration on MRS agar followed by aerobic incubation at 37 °C for 24 h. The LAB concentration administered in the present study was selected based off previous work [19].

2.3. Bifidobacteria

In previous work (unpublished), our team isolated the two B. saeculare isolates (B2-2, B3-4) used in these trials, which were recovered from cecal droppings collected from broody turkey hens. These samples were enriched in Trypticase Phytone Yeast Extract (TPY) broth and incubated anaerobically on an orbital shaker at 37 °C for 48 h [26,27]. After enrichment, samples were struck for isolation on a TOS-propionate (TOS Agar, EMD Millipore VM795943) agar containing 440 mg/L mupirocin (MUP, Glemark Pharmaceuticals NDC 68,462-180-22), as previously described [28,29]. TOS + MUP agar plates were incubated anaerobically using a BD GasPak EZ Anaerobe Container System (VWR, Suwanee, GA, BD 260678) at 37 °C for 48 h. Next, distinct individual colonies were removed and used to inoculate fresh TPY broth for secondary enrichment. Samples were incubated anaerobically on an orbital shaker at 37 °C for 48 h. Similar to above, turbid TPY broth cultures were streaked onto TOS + MUP following the secondary enrichment and incubated anaerobically at 37 °C for 48 h. An individual colony from each plate was observed microscopically using a bright-field microscope to confirm Bifidobacterium morphology. Ten candidates were isolated and consisted of five B. saeculare and five B. pseudolongum. Identification was confirmed by 16 S sequencing. Since bifidobacteria are obligate anaerobes, each candidate was evaluated to determine the resilience of the bacteria when exposed to oxygen in vitro. A 200 µL sample of a turbid culture was placed in a 96 well plate, diluted, and plated on TPY and TOS agar at times 0, 30, 60, and 120 min. Two B. saeculare (B2-2 and B3-4) isolates were selected based on their ability to persist when exposed to oxygen for 120 min (unpublished data). Further evaluation of B2-2 and B3-4 indicated that these candidates readily colonized the gut of chicks at hatch without negatively impacting hatchability when administered by an in ovo injection (1 × 10⁸ CFU/200 uL/embryo) at DOE18 (unpublished data).

For the current study, a stock aliquot of B2-2 and B3-4 was thawed and swabbed onto two TOS + MUP agar to generate a ‘lawn-of-growth’. Plates were incubated anaerobically at 37 °C for 48 h. Following incubation, sterile cotton swabs were used to gently scrape colonies from the agar plates for inoculating an additional 20 TOS + MUP agar plates. After anaerobic incubation at 37 °C for 48 h, 5 mL of 0.9% sterile saline was added to each plate, and a sterile cell plate scraper was used to gently remove the colonies from the surface of the agar. A serological pipet was used to collect the cell suspension and transfer it to a 50 mL conical tube. This process was repeated for all plates for each isolate. The cell suspension was washed by centrifugation at 1800× g for 15 min. Following centrifugation, the supernatant was decanted, and the pellet was resuspended in 20 mL of sterile saline to remove any potential residual MUP. The final bifidobacteria concentration for in ovo treatments was 1 × 10⁸ CFU/embryo based on our previous unpublished work. Treatments were plated onto TOS + MUP plates and incubated anaerobically at 37 °C for 48 h to confirm the final concentration for each trial.

2.4. Application of Probiotic Treatments

To administer the probiotic treatments to DOE18 embryos, a sterile ½ inch 18-gauge needle was used to carefully puncture the surface of the eggshell where the air cell was located. Following this, 200 μL of the respective treatment was administered into the amniotic cavity using a sterile stainless steel 21-gauge blunt tip needle and 1 mL syringe. The embryos were then placed into the respective hatchers but remained in the transport flats until challenge on DOE19. Treatment concentrations were confirmed as described above. NC did not receive any treatment nor application of the vehicle.

2.5. Challenge Preparation and Application

Preparation and application of the pathogen mix (PM) followed the procedures described by Graham et al. [24]. In the current study, modifications were made to the composition of the PM challenge. Specifically, the PM challenge consisted of presumptive pathogens commonly recovered from commercial poultry production, including two wild-type Escherichia coli isolates (LG 2016 field isolate and 021 hatchery isolate), Enterococcus faecalis (031 B hatchery isolate), and Enterococcus cecorum (EC7 field isolate). E. cecorum had not been included in the PM challenge in previous studies [24,30]. EC7 used in the current study has been shown to reduce early performance in broiler chickens exposed during late embryogenesis [31]. No further attempts were made to determine the virulence profile, etc., of the isolates used for the challenge. All challenge isolates were grown in 500 mL of TSB and prepared as previously described [24,30]. In brief, the E. coli isolates were grown aerobically, and the Enterococcus spp. isolates were grown in microaerophilic conditions (5% CO₂). Following incubation, each turbid culture was concentrated by centrifugation at 1 × at 1800× g for 15 min. Pellets were resuspended in ~50 mL of 0.9% sterile saline and plated on respective media to determine the CFU/mL. Cultures were held at 4 C overnight. At DOE19, the appropriate volume of each washed culture for the PM challenge was removed and concentrated by centrifugation. All pellets were resuspended in 2 × tryptic soy broth (TSB, cat. no. 90,000-378, VWR, Suwanee, GA, USA) containing 0.01% xanthan gum serving as a nutrient source and thickening agent [24]. After combining and mixing the PM challenge, a subsample of the PM was removed and enumerated using the drop plate method on MacConkey Agar (VWR, Suwanee, GA, USA, Cat. No. 89,405-630) and CHROMagar Orientation (DRG International, Springfield, NJ, USA, RT412, CO agar) to confirm the CFU/mL. For all trials, the concentrations for each isolate included in PM was confirmed as ~1 × 10⁹ CFU/mL or ~1 × 10⁷ CFU/100 μL. As described by Graham et al. (2022) [24], embryos were removed from the hatchers by treatment at DOE19, and 100 μL of the PM challenge was applied over a 28 mm area of the air cell (excluding NC). Attention was given to not spreading the challenge inoculum directly over the pilot hole made on the surface of the eggshell for the in ovo injection of probiotic treatments. After the PM application, embryos were removed from the transport trays and placed into the hatcher trays.

2.6. Gastrointestinal Sampling

For all trials, gut samples, duodenum to cloaca, were aseptically removed from n = 12 chicks/treatment on DOE21 (or DOH) and collected into sterile bags. Gut samples were weighed and homogenized, and 1:4 wt/vol dilutions were made using sterile 0.9% saline. Ten-fold dilutions of each sample were made in sterile 96-well plates followed by drop plating of each dilution to evaluate presumptive Gram-negative bacteria, LAB, Bifidobacterium spp., and Enterococcus spp. on the media described above. MacConkey, MRS, and CO agar plates were incubated at 37 °C for 18–24 h. MacConkey and MRS plates were incubated aerobically, and CO plates were incubated in 5% CO₂. TOS + MUP plates were incubated aerobically at 37 °C for 48 h. Colonies were counted for each plate and recovery is expressed as Log10 CFU/g of sample.

2.7. Animal Source

All trials utilized candled, unvaccinated eighteen-d-old Ross 308 embryos that were purchased from a local commercial hatchery (George’s, Inc. Hatchery; Springdale, AR, USA). For each trial, embryos were obtained at DOE18 from the same hatchery and source flock and transferred to the University of Arkansas Poultry Health Laboratory facility. At DO18, eggs were randomly allocated and placed into separate hatchers based on the treatment group.

2.8. Statistical Analysis

Microbial recovery from DOH gut samples and performance data were subjected to one-way ANOVA using JMP Pro 17. Means were further separated using Student’s t-test. Significance reported as p < 0.05 unless otherwise noted. DOH gut recovery is expressed as mean Log10 CFU/g presented by individual trials or as a meta-analysis by treatment for trials 2–4. For mortality and adjusted hatchability, each chick served as an experimental unit, and chi-square (JMP 17 Pro) was used to determine significant differences between NC and treatment groups. For BW, BWG, and FCR metrics, the pen served as the experimental unit, whereas individual animals served as the experimental unit for microbial recovery.

3. Results

3.1. DOH Gut Recovery

Microbial recovery from gut samples collected at DOH is presented in

Table 1. Bacterial recovery (Log10 CFU/g) from gut samples at hatch by trial.

Trial	Treatment 1	Gram-Negative Bacteria	Bifidobacterium spp.	Lactic Acid Bacteria	Enterococcus spp.
Trial 1	NC	1.64 ± 0.62 cde	0.00 ± 0.00 c	0.98 ± 0.70 d	0.00 ± 0.00 e
	PM	1.32 ± 0.75 cde	0.22 ± 0.22 bc	2.61 ± 0.89 cd	0.64 ± 0.64 de
	LAB18	3.82 ± 0.68 b	0.73 ± 0.39 abc	4.94 ± 0.70 ab	2.36 ± 0.90 cd
	LAB46	0.99 ± 0.69 de	0.28 ± 0.28 bc	4.65 ± 0.56 ab	1.90 ± 0.84 cde
	B2-2	2.47 ± 0.82 bcd	0.53 ± 0.53 bc	5.03 ± 0.74 ab	5.38 ± 0.81 ab
	B3-4	3.18 ± 0.86 bc	1.49 ± 0.69 ab	6.57 ± 0.34 a	6.05 ± 0.66 a
	LAB18 + B2-2	5.93 ± 0.63 a	1.23 ± 0.83 abc	5.88 ± 0.64 a	1.87 ± 0.98 cde
	LAB46 + B3-4	0.24 ± 0.24 e	2.13 ± 0.71 a	3.07 ± 0.70 bc	3.65 ± 0.99 bc
	LAB18 + B3-4	1.61 ± 0.71 cde	0.00 ± 0.00 c	3.08 ± 0.85 bc	0.24 ± 0.24 de
	LAB46 + B2-2	4.40 ± 0.88 ab	1.10 ± 0.74 abc	2.39 ± 0.80 cd	2.35 ± 0.86 cd
Trial 2	NC	0.22 ± 0.22 b	0.00 ± 0.00 c	0.00 ± 0.00 c	0.00 ± 0.00 c
	PM	2.77 ± 0.84 a	0.00 ± 0.00 c	0.00 ± 0.00 c	4.11 ± 0.86 a
	LAB46	2.63 ± 0.88 a	0.00 ± 0.00 c	5.57 ± 0.58 a	1.86 ± 0.69 bc
	LAB46 + B2-2	2.28 ± 0.98 ab	2.81 ± 0.92 b	3.49 ± 0.88 b	1.81 ± 0.84 bc
	LAB46 + B3-4	2.64 ± 0.90 a	1.16 ± 0.64 c	5.75 ± 0.63 a	3.57 ± 0.86 ab
	LAB46 + B-Combo	3.09 ± 0.86 a	5.79 ± 0.58 a	4.60 ± 0.26 ab	1.43 ± 0.57 c
Trial 3	NC	0.00 ± 0.00 c	0.00 ± 0.00 b	0.00 ± 0.00 b	0.00 ± 0.00 c
	PM	3.51 ± 0.83 b	0.00 ± 0.00 b	0.84 ± 0.45 b	4.75 ± 0.69 a
	LAB46	3.96 ± 0.89 b	0.00 ± 0.00 b	5.45 ± 0.58 a	2.76 ± 0.89 ab
	LAB46 + B2-2	0.38 ± 0.38 c	1.92 ± 0.74 a	5.14 ± 0.54 a	4.24 ± 0.72 a
	LAB46 + B3-4	6.11 ± 0.69 a	3.01 ± 0.81 a	4.97 ± 0.71 a	1.10 ± 0.75 bc
	LAB46 + B-Combo	2.57 ± 1.11 b	2.67 ± 0.76 a	5.82 ± 0.37 a	2.16 ± 0.84 b
Trial 4	NC	0.00 ± 0.00 c	1.04 ± 0.70 b	0.31 ± 0.31 c	0.87 ± 0.61 d
	PM	7.05 ± 0.42 a	0.31 ± 0.31 b	4.38 ± 0.99 b	6.45 ± 0.49 a
	LAB46	6.26 ± 0.57 a	0.00 ± 0.00 b	5.52 ± 0.68 ab	3.24 ± 0.91 c
	LAB46 + B2-2	2.60 ± 0.77 b	5.10 ± 0.84 a	6.26 ± 0.38 a	4.17 ± 1.01 bc
	LAB46 + B3-4	5.64 ± 1.21 a	3.50 ± 1.08 a	7.08 ± 0.70 a	5.87 ± 0.92 ab
	LAB46 + B-Combo	6.55 ± 0.56 a	5.26 ± 0.97 a	5.32 ± 0.60 ab	6.27 ± 0.67 ab

Data reported as mean ± SE, n = 12/treatment. ^a,b,c,d,e Superscripts indicate significance across treatment groups by trial and column at p < 0.05. ¹ Treatment abbreviations: NC (Negative Control), PM (Pathogen Mix), LAB18 (LAB isolate 18), LAB46 (LAB isolate 46), B2-2 (Bifidobacterium saeculare isolate 2–2), B3-4 (B. saeculare isolate 3–4), LAB46 + B-Combo (LAB46 + B2-2 + B3-4).

. In trial one, LAB46 and LAB46 + B3-4 had the lowest recovery of Gram-negative bacteria from the gut compared to all groups. LAB18 and LAB18 + B2-2 had markedly more Gram-negative bacterial recovery compared to PM. However, none of the groups were significantly (p > 0.05) different from PM. All other probiotic treatments resulted in numerically higher, and in some cases significantly higher, Gram-negative bacterial recovery than PM. In trial two, all groups were statistically similar to PM for Gram-negative bacterial recovery from the gut, excluding NC, which was not significantly different from LAB46 + B2-2. In trial three, Gram-negative bacterial recovery for PM, LAB46, and LAB46 + B-Combo was similar, and all three were significantly higher than LAB46 + B2-2 and NC, while LAB46 + B3-4 was significantly higher than all groups. In trial four, LAB46 + B2-2 had significantly lower Gram-negative bacterial recovery compared to PM, while LAB46, LAB46 + B3-4, and LAB46 + B-Combo were not significantly different from PM.

In trial one, Bifidobacterium spp. recovery from the gut at DOH was increased in groups treated with B. saeculare compared to NC, PM, and groups only treated with LAB. However, no bifidobacteria was recovered from LAB18 + B3-4-treated chicks. In trial two, LAB46 + B2-2- and LAB46 + B-Combo-treated chicks had significantly higher Bifidobacterium recovery than all other groups, while recovery from LAB46 + B3-4 was not different to NC, PM, and LAB46. In trials three and four, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo had significantly higher bifidobacteria recovery from the gut at hatch compared to NC, PM, and LAB46.

LAB recovery from the gut at DOH in trial one was significantly increased across all treatment groups compared to NC and PM, except LAB46 + B3-4, LAB18 + B3-4, and LAB46 + B2-2. In trials two and three, LAB recovery was markedly elevated in the gut at DOH for LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo compared to NC and PM. A similar trend was observed in trial four.

In trial one, in ovo treatments did not reduce Enterococcus spp. recovery from DOH gut compared to PM. In trial two, NC, LAB46, LAB46 + B2-2, and LAB46 + B-Combo had significantly lower Enterococcus spp. recovery from the gut at DOH compared to PM. Although LAB46 + B3-4 had numerically lower Enterococcus spp. recovery compared to PM in trial two, this difference was not significant. In trial three, Enterococcus spp. recovery from the gut was significantly lower in NC, LAB46 + B3-4, and LAB46 + B-Combo compared to PM, while recovery was only numerically lower for LAB46 and LAB46 + B2-2 compared to PM. In trial four, NC, LAB46, and LAB46 + B2-2 had markedly less Enterococcus spp. recovered from the gut at hatch compared to PM.

3.2. Meta-Analysis of DOH Gut–Trials 2–4

A meta-analysis of bacterial recovery from the gut at DOH for trials two, three, and four is presented in Figure 1. NC and LAB46 + B2-2 were the only treatments that resulted in a significant reduction in Gram-negative bacterial recovery from DOH gut samples compared to PM (Figure 1). LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo significantly reduced Enterococcus spp. recovery compared to PM, while recovery from NC was significantly lower than all treatments. Groups containing LAB resulted in a significant increase in LAB compared to NC and PM. Likewise, groups containing B. saeculare had a significant increase in Bifidobacterium spp. recovered from the gut collected at hatch compared to NC and PM.

3.3. Performance

No significant differences were observed for hatchability (

Table 2. Adjusted hatchability (Trials 2, 3, and 4).

Treatment 1	Trial 2	Trial 3	Trial 4
NC	237/239 (99.16)	357/354 (99.16)	432/441 (97.96)
PM	230/236 (97.46)	356/351 (98.60)	439/446 (98.43)
LAB46	237/238 (99.58)	358/357 (99.71)	221/223 (99.10)
LAB46 + B3-4	215/219 (98.17)	359/352 (98.06)	208/209 (99.52)
LAB46 + B2-2	230/230 (100)	357/353 (98.88)	206/207 (99.52)
LAB46 + B-Combo	224/225 (99.56)	354/352 (99.42)	206/208 (99.04)

Adjusted hatchability excluded embryos not viable prior to DOE18 and infertile embryos. No significant differences were detected for adjusted hatchability (chi-square test p > 0.05). ¹ Treatment abbreviations: NC (Negative Control), PM (Pathogen Mix), LAB46 (LAB isolate 46), B2-2 (Bifidobacterium saeculare isolate 2–2), B3-4 (B. saeculare isolate 3–4), LAB46 + B-Combo (LAB46 + B2-2 + B3-4).

). Average BW, BWG, FCR, and mortality results for trials two and four are presented in

Table 3. Average body weight (BW), body weight gain (BWG), feed conversion ratio (FCR), and mortality (%) (Trial 2 and 4).

Trial	Treatment 1	BW (g)			BWG (g)		FCR		Mortality (%)
		d0	d7	d14	d0–7	d0–14	d0–7	d0–14	d0–7	d0–14
Trial 2	NC	38.5 ± 0.02 c	143.4 ± 1.97	394.1 ± 7.41	103.85 ± 1.85	398.83 ± 5.63 ab	1.27 ± 0.04 b	1.37 ± 0.03	1/160 (0.63)	2/160 (1.25)
	PM	38.6 ± 0.06 c	144.0 ± 2.67	358.9 ± 10.40	105.46 ± 2.65	375.83 ± 10.27 b	1.41 ± 0.10 ab	1.55 ± 0.11	0/160 (0)	0/160 (0)
	LAB46	38.5 ± 0.02 c	148.9 ± 4.44	399.5 ± 4.13	110.34 ± 4.44	417.22 ± 3.70 a	1.30 ± 0.03 ab	1.32 ± 0.02	0/160 (0)	0/160 (0)
	LAB46 + B2-2	39.3 ± 0.06 a	141.8 ± 5.88	400.2 ± 17.53	102.54 ± 5.85	415.97 ± 17.60 a	1.53 ± 0.15 a	1.53 ± 0.21	0/160 (0)	1/160 (0.63)
	LAB46 + B3-4	38.8 ± 0.04 b	146.8 ± 1.55	387.4 ± 12.03	107.99 ± 1.56	404.39 ± 12.27 ab	1.33 ± 0.06 ab	1.39 ± 0.03	0/160 (0)	0/160 (0)
	LAB46 + B-Combo	38.5 ± 0.04 c	148.3 ± 2.52	389.3 ± 12.92	108.94 ± 2.69	405.76 ± 13.29 a	1.38 ± 0.04 ab	1.39 ± 0.04	1/160 (0.63)	1/160 (0.63)
Trial 4	NC	38.7 ± 0.04 ab	133.4 ± 3.21 ab	346.5 ± 9.31	90.86 ± 3.21	299.00 ± 9.39	1.56 ± 0.10	1.74 ± 0.10	4/264 (1.52)	4/264 (1.52)
	PM	38.6 ± 0.06 b	134.0 ± 3.26 b	344.9 ± 12.73	91.37 ± 3.21	296.80 ± 12.58	1.54 ± 0.09	1.64 ± 0.06	1/242 (0.41)	4/242 (1.65)
	LAB46	38.5 ± 0.06 b	140.1 ± 3.62 ab	360.6 ± 9.42	97.70 ± 3.59	312.40 ± 9.64	1.49 ± 0.11	1.62 ± 0.09	1/198 (0.51)	4/198 (2.02)
	LAB46 + B2-2	38.5 ± 0.06 b	142.1 ± 5.14 ab	361.8 ± 15.88	99.81 ± 5.12	314.80 ± 15.86	1.30 ± 0.07	1.51 ± 0.09	0/154 (0)	0/154 (0)
	LAB46 + B3-4	38.5 ± 0.04 b	140.1 ± 1.51 ab	363.3 ± 1.18	95.36 ± 2.49	314.60 ± 1.74	1.50 ± 0.11	1.63 ± 0.09	3/132 (2.27)	5/132 (3.79)
	LAB46 + B-Combo	38.9 ± 0.25 a	144.3 ± 4.44 a	365.2 ± 11.38	100.10 ± 4.60	316.80 ± 11.15	1.46 ± 0.10	1.63 ± 0.09	4/154 (2.60)	5/154 (3.25)

^a,b,c Superscripts indicate significant difference across treatment groups by trial and column at p < 0.05. Trial 2 n = 8 pens/treatment, n = 20 chicks/pen. Trial 4 n = 6–12 pens/treatment, n = 22 chicks/pen. Mortality reported as a percentage of total mortality/total chicks placed by period. Chi-square test used to determine significant differences at p < 0.05 for mortality only. ¹ Treatment abbreviations: NC (Negative Control), PM (Pathogen Mix), LAB46 (LAB isolate 46), B2-2 (Bifidobacterium saeculare isolate 2–2), B3-4 (B. saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + B2-2 + B3-4).

. While significant differences in DOH average BW were observed between groups in both trials, the average treatment BW in both trials was 0.8 g or less across all groups. No significant differences were observed for average BW at d7 or d14 in trial two. In trial four, the average BW at d7 was significantly higher in LAB46 + B-Combo compared to PM, but no significant differences were observed in BW at d14. In trial two, average 0–14 d BWG was markedly improved in LAB46, LAB46 + B2-2, and LAB46 + B-Combo compared to PM. In trial four, although a 20 g improvement in 0–14 d BWG was observed for LAB46 + B-Combo compared to PM, there were no significant differences between any groups. No significant differences were observed in mortality for either trial. FCR was numerically improved in LAB46, LAB46 + 2-2, and LAB46 + B-Combo compared to PM in both trials.

4. Discussion

The artificial introduction of defined probiotic cultures or undefined competitive exclusion products as early as possible is advantageous to prevent colonization by hatchery-related microorganisms and to promote the early establishment of gut microflora. Pre-hatch probiotic application methods have included in ovo or spray applications to the eggshells. In ovo application of an undefined competitive exclusion culture at DOE17 translated to reduced Salmonella colonization in chicks post-hatch compared to non-treated chicks [32]. However, the exact mechanisms of action of the competitive exclusion culture remain unknown. In contrast, there has been a heavy emphasis on utilizing beneficial Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., or Bacillus spp. in defined probiotic products for pre- and post-hatch application because of efficacy and production feasibility. These beneficial bacteria may inhibit pathogens by reducing the environmental pH in the gastrointestinal tract, which creates an unfavorable environment for pathogenic microorganisms [33], or by producing bacteriocins that exude antimicrobial properties [34]. Studies evaluating the effect of in ovo application of defined probiotic bacterial cultures on enteric colonization by beneficial bacterial populations and exclusion of pathogens post-hatch have also been conducted [10,19,35]. Teague et al. demonstrated that the in ovo administration of FloraMax-B11, a commercially available probiotic containing lactic acid bacteria including LAB18 and LAB46 used in the present study, on DOE18 reduced Gram-negative bacteria in the gut at hatch and effectively reduced Salmonella Enteritidis colonization in the ceca [35]. In another study, in ovo application of E. faecium or B. subtilis into the amniotic cavity at DOE17.5 was associated with reduced Salmonella colonization in the ceca of chickens at 17 days of age [35]. In another study, in ovo administration of L. plantarum at DOE 18.5 reduced Gram-negative bacteria in the gut at 14 days of age [36]. Bifidobacterium bifidum, B. animalis, B. longum, or B. infantis application via in ovo injection into the yolk sac at DOE17 also translated to increased Lactobacillus spp. and Bifidobacterium spp. populations in the gut at hatch but lower Gram-negative bacterial recovery from the gut at 28 days-of-age [10]. These studies suggest embryonic exposure to beneficial pioneer colonizers of the gastrointestinal tract may be an effective method to readily exclude colonization by opportunistic and pathogenic microorganisms post-hatch. It is important to note that hatchability was not reported [10] but was negatively affected in another experiment conducted by the same team, where 10⁷ or 10⁹ CFU/egg of B. bifidum or B. longum was in ovo injected into the yolk sac at DOE17 [10]. In contrast, the hatchability of fertile eggs was not impacted by 10⁶ CFU/egg B. animalis injected into the amnionic cavity [37], which was similar to our observations in the present study, where administration of B. saeculare at 10⁸ CFU/egg alone or in combination with lactic acid bacteria into the amniotic cavity at DOE18 did not impact late embryonic development or hatchability. Thus, microorganisms, dose, DOE, and the site of administration are all critical factors that must be considered when investigating the efficacy of in-ovo-applied probiotics.

To our knowledge, B. saeculare has been minimally tested in chickens. B. saeculare has been recovered from rabbit feces [38] and identified in cecal contents obtained from chickens [39,40,41]. There is a commercial product, AVIGUARD, which contains 23 anaerobic bacteria, including one strain of B. saeculare [40]. Metabolic profiles in the ceca of chickens treated with AVIGUARD at hatch were shown to be different than the profile of non-treated controls [40]. More research is needed to elucidate which bacteria or combination of bacteria in commercial products causes these fluctuations in the cecal metabolic profile. There is evidence that the administration of multiple beneficial anaerobic bacteria, such as Bifidobacterium spp., is necessary to have true competitive exclusion effects in the gastrointestinal tract of chickens [41,42]. Synergy between lactic acid bacteria and/or bifidobacteria administered by in ovo injections into the amniotic cavity at DOE18 has been reported [35,43,44]. Since research on B. saeculare specifically has been lacking in poultry, a more concerted effort to assess B. saeculare alone or in combination with LAB 18 or LAB46 was of interest.

The present study serves as one of the first described attempts to combine B. saeculare strains isolated from a broody hen with LAB18 or LAB46. An environmental contamination model was used in a laboratory setting to create microbial contamination commonly associated with commercial hatch cabinets. This model was employed to evaluate the ability of pioneer colonizers, such as LAB and/or B. saeculare, to prevent enteric colonization by opportunistic bacteria during the hatching phase and to determine if exposure to the beneficial bacteria at DOE18 improved 7- and 14-day performance in broiler chickens. The in ovo administration of LAB or bifidobacteria increased relevant populations in the gut at hatch, indicating that the probiotic dose and administration route were effective. Performance was not evaluated for LAB18 alone or in combination with bifidobacteria due to enteric recovery in trial one. However, in trials two and four, a consistent reduction in Gram-negative bacteria was observed for chicks that received LAB46 + B2-2 at DOE18. Performance improvements, such as improved 0–14-day BWG, were associated with pre-hatch exposure to LAB46, LAB46 + B2-2, and LAB46 + B-Combo. Additionally, day-seven BW was improved for LAB46 + B-Combo in trial four. This indicates that introducing beneficial bacteria during the pre-hatch phase may prevent or perturb colonization by Gram-negative bacteria and that these effects, including those related to performance, may vary based on the probiotic isolate(s) used. It is important to note that cross-inhibition assays between the LAB and B. saeculare strains used in this study were not conducted. The reduced efficacy of LAB18 and B2-2 or B3-4 combinations, in terms of perturbing colonization by Gram-negative bacteria or Enterococcus spp. during the hatching phase, could be related to inhibitory factors, such as bacteriocins, and will be explored in future work.

5. Conclusions

The results from the current study suggest that the pre-hatch application of combinations of B. saeculare and lactic acid bacteria could be used to promote early colonization by beneficial bacteria and improve performance in chickens. Isolates and combinations thereof should be carefully selected since additive or synergistic effects do not always occur. Furthermore, the effect of in-ovo-applied probiotic treatments on gut colonization at hatch may not align with performance observations, highlighting the complexity of gut development, immunomodulation, and succession of the microbiome. Additional research is ongoing to assess the effects of LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo treatments on these parameters.

6. Patents

Danielle Graham, Mitchell Rowland, Billy M. Hargis, and Kyle Teague, Method to Reduce Enteric Colonization by Hatchery-Associated Opportunistic Pathogens and Improve Performance of Chickens, PCT Application No. PCT/US2024/056418 (filed 18 November 2024).