Biophysiology of in ovo administered bioactive substances to improve gastrointestinal tract development, mucosal immunity, and microbiota in broiler chicks

Abstract

Early embryonic exogenous feeding of bioactive substances is a topic of interest in poultry production, potentially improving gastrointestinal tract (GIT) development, stimulating immunization, and maximizing the protection capability of newly hatched chicks. However, the biophysiological actions and effects of in ovo administered bioactive substances are inconsistent or not fully understood. Thus, this paper summarizes the functional effects of bioactive substances and their interaction merits to augment GIT development, the immune system, and microbial homeostasis in newly hatched chicks. Prebiotics, probiotics, and synbiotics are potential bioactive substances that have been administered in embryonic eggs. Their biological effects are enhanced by a variety of mechanisms, including the production of antimicrobial peptides and antibiotic responses, regulation of T lymphocyte numbers and immune-related genes in either up- or downregulation fashion, and enhancement of macrophage phagocytic capacity. These actions occur directly through the interaction with immune cell receptors, stimulation of endocytosis, and phagocytosis. The underlying mechanisms of bioactive substance activity are multifaceted, enhancing GIT development, and improving both the innate and adaptive immune systems. Thus summarizing these modes of action of prebiotics, probiotics and synbiotics can result in more informed decisions and also provides baseline for further research.

INTRODUCTION

In commercial broilers, more than 50% of the productive lifespan of chickens is determined by the conditions of incubation and neonatal periods (Ferket, 2012; Patricia et al., 2020; Kouassi and Monika, 2023). The time period from the 18th day of incubation (DOI) to 4-days posthatch is considered the critical period for rapid intestinal development (Dibner et al., 1996; Iji et al., 2001a), and the survival and growth of chicks (Ferket, 2006). During this critical period, chicks also undergo both metabolic and physiological shifts from endogenous nutrients to exogenous feed utilization (Iji et al., 2001a; De Oliveira et al., 2008; Ferket, 2012; Patricia et al., 2020). This transition enhances high energy and nutrient demand, potentially, leading to an imbalance of nutrients or malnutrition (Kadam et al., 2013; Ghanaatparast-Rashti et al., 2018; Patricia et al., 2020); and limited development of embryos and posthatch growth performance (Ohta et al., 1999), which can hinders the development and maturation of the intestine of chicks (Geyra et al., 2001a; Gao et al., 2017a).

In addition, the immune system of neonatal chick is also immature and inefficient. Although many authors have indicated that most of the development of the immune system is complete at the late embryonic phase (Bar-Shira and Friedman, 2006; Reemers et al., 2010; Eren et al., 2016; Song et al., 2021), the maturation and response of the immune system increase with posthatch age until 30 to 34 d (Song et al., 2021). Thus, chicks are highly vulnerable to environmental threats during the first weeks of posthatching (Farnell et al., 2006; Pender et al., 2017).

For these above aforementioned reasons and banning of antibiotic growth promoters (AGP), in ovo feeding of bioactive substances has been studied regard to its impact on GIT function and the micorbiome profile (De Cesare et al., 2019), it has been found that to correct nutrient imbalance (Foye et al., 2006; Nasir and Peebles, 2018), improve growth rate and feed conversion efficiency, and enhance weight (Bogucka et al., 2017; Stefaniak et al., 2019; Reicher et al., 2022), development and maturation of the immune system (Murate et al., 2015; Pender et al., 2017; Stefaniak et al., 2019; Qamar et al., 2020), and reduce the rate and severity of enteric infections (Dibner et al., 1998; Song et al., 2021). In ovo administration of bioactive substances has also been shown to improve intestinal protection, antioxidant capacity and apoptosis (Bai et al., 2013; Broom and Kogut, 2018; Wu et al., 2019).

In this context, the in ovo feeding technique has shown promising and has become popular to ensure access to bioactive substances and nutrients at the embryonic development stage of chicks (BogusLAwska-Tryk et al., 2012; Cox and Dalloul, 2015; Roto et al., 2016; Gao et al., 2017b; Ghanaatparast-Rashti et al., 2018; Nasir and Peebles, 2018; Siwek et al., 2018; Das et al., 2021). The method was created in the 1980s to administer the Marek's disease vaccine, and resulted chicks with significantly better immunity, performance, and gut health (Sharma and Burmester, 1982; Siwek et al., 2018). The earliest in ovo bioactive substance demonstration attempts have shown promising results, with long lived biological effects (Siwek et al., 2018; Patricia et al., 2020; Dunislawska et al., 2021).

The bioactive substances that have been used in broiler production through in ovo delivery include prebiotics, probiotics, synbiotics, amino acids, carbohydrates, hormones, CpG oligodeoxynucleotides, and proteins (Saeed et al., 2018b; Arif et al., 2019; Pirzado et al., 2021a; Kouassi and Monika, 2023). In recent years, the in ovo application of prebiotics, probiotics, and synbiotics substances has garnered considerable attention in poultry production due to its potential to revolutionize performance and health in avian species (Brisbin et al., 2008; Kouassi and Monika, 2023). However, challenges persist in implementing these strategies, including optimal dosage determination (Bednarczyk et al., 2016; Dankowiakowska et al., 2019), consideration of the placement site (Villaluenga et al., 2004; BogusLAwska-Tryk et al., 2012; Bednarczyk et al., 2016; Limsay, 2018; Dunislawska et al., 2021; Shehata et al., 2021; Kouassi and Monika, 2023), injection timing (Shehata et al., 2021), probiotic strain selection, and compatibility with commercial production systems. Moreover, inconsistencies in study outcomes emphasize the need for standardized protocols and comprehensive understanding of host–microbiota interactions to ensure consistent benefits across various conditions and poultry breeds (Pourabedin and Zhao, 2015).

Negative results in terms of hatchability, performance, and mortality have also been noted. These discrepancies might be due to the bioactivity mechanism and their effectiveness may be closely linked with the structure and composition of the used bioactive substance (Wassie et al., 2021). Information on the modulator action of bioactive substances on intestinal development, the immune system, and the GIT micorbiomes is inconsistent and therefore requires further insight. Thus, the intention of this review is to summarize the modulation mechanism of prebiotics, probiotics, and synbiotics on the morphology of the GIT, mucosal immunity, microbiota, and pathogen combating capability in broiler chicks.

INTESTINAL DEVELOPMENT AND FUNCTION

The successful development of numerous intricate and highly specialized sections of GIT is depends on the availability of nutrients in the egg during the embryonic and posthatching stages of chicks (Sobolewska et al., 2017b; Kouassi and Monika, 2023). However, endogenous nutrients alone may not be sufficient to sustain the late stage of embryonic development and the hatching process. Numerous previous studies have confirmed that the in ovo delivery of prebiotics, probiotics, and synbiotics have ameliorative modulation effects on GIT development in chicks (Bogucka et al., 2017; Kouassi and Monika, 2023) (Table 1). For instance, Mista et al. (2017) demonstrated that in ovo injection of L. lactis subsp. lactis IBB (1,000 CFU) and insulin (1.76 mg/embryo) combination (synbiotics) at 12 DOI improved intestinal morphology, cecal SCFA profile, and the growth of broiler chickens. Consistently, in ovo feeding (IOF) of different amino acids (AAs) at the late embryonic phase has been shown remarkable to improve the morphological and functional development of the intestinal mucosa (Al-Murrani, 1982; Gao et al., 2017b; Nazem et al., 2017). Likewise, the weight of the small intestine at 4 and 21 DOI was linearly changed with the inclusion of sodium butyrate during posthatch feeding (Lan et al., 2020), and 10% degraded date pits supplementation also changed the intestine of broilers (Alyileili et al., 2020).

Table 1.

The effect of in ovo administered bioactive substances on intestinal development of broiler.

Bioactive substance (pre, pro, and synbiotic)	Level or doze	Administration time	Injection site	Ameliorative results or function	References
L-Arg + Rj	1% +20%	18 DOI	Amniotic fluid	Increase relative intestine weight and Villi measurements (gut development)	Hassan et al., 2018
NAG	1.5 mg	17.5 DOI	Amniotic fluid	Increase the density of goblet cells Decrease CD, Increase VH and VH:CD ratio	Wang et al., 2020
MOS	0.1%	12 or 17 DOI	Amnion fluid	Increase intestinal thickness Increase VH	Cheled-Shoval et al., 2011; Berrocoso et al., 2017; Reicher et al., 2022
β-Hydroxy-β-methyl butyrate, dextrin, maltose, sucrose	1 g/L, 200 g/L, 25 g/L, and 25 g/L, respectively	17 DOI	Amniotic fluid	Increase small intestine weight Increase the size of villus Increase intestinal digestion capacity	Tako et al., 2004
B2tos	0.528 mg	12 or 17 DOI	Amnion fluid	Increase villus surface area and height	Reicher et al., 2022
Raffinose	4.5 mg	12 or 17 DOI	Amnion fluid	Increase VH:CD ratio	Berrocoso et al., 2017
Astragalus polysaccharide (APS)	1 mg and 2 mg	18 DOI	Amnion fluid	Increase VH Increase CD Increase VH/CD ratio	Yang et al., 2021
Xylotetraose	3 mg	17 DOI	Amnion fluid	Increase VH/CD ratio	Amit et al., 2022
Inulin	1.76 mg	12 or 17 DOI	Amnion fluid	Increase villus surface area	Reicher et al., 2022
Extract of Laminaria species of seaweed	0.88 mg	12 DOI	Air sac	Enhance development of duodenum	Sobolewska et al., 2017a
Lactobacillus salivarius and L. plantarum	109 CFU	17.5 DOI	Air sac	Increase VH of jejunum	Khaligh et al., 2018
Bifidobacterium bifidum and Bifidobacterium longum	1 × 107 - 5 × 109 CFU	17 DOI	Yolk sac	Significant improve VH	El-Moneim et al., 2020
Inulin, GOS, inulin + lactis subsp. Lactis, and GOS + L. lactis subsp. Cremoris	1.76 mg, 0.528 mg, inulin +1,000 CFU, and GOS + 1,000 CFU, respectively	12 DOI	Air sac	Improve the cecal SCFA profile and intestinal morphology	Mista et al., 2017
RFO	1.5−4.5 mg	12 DOI	Air sac	Improves ileum mucosa morphology	Berrocoso et al., 2017
DiNovo (prebiotics)	0.88 mg	12 DOI	Air sac	Improves histomorphology of intestine	Sobolewska et al., 2017b

This table shows that the efficacy of in ovo injection bioactive substances, which varies on time of injection, site of injection, dosage level. DOI, days of incubation; RFO, raffinose family oligosaccharide; NAG, N-acetyl-L-glutamate; Rj, Royal Jelly; MOS, mannan oligosaccharide; B2tos, transgalacto-oligosaccharide.

Immediately after hatching, the proportional growth of the small intestine is greater than the body weight (BW) of chicks; peaks within 6 to 10 d of hatching (DOH) (Katanbaf et al., 1988; Sklan, 2001) and is completely formed by 12 DOH (Alcantara et al., 2013): this might be due to the accelerated processes of enterocyte proliferation and differentiation (Geyra et al., 2001a). In a previous study, the relative lengths of the duodenum (21 DOH), jejunum (14 and 21 DOH), ileum (14 and 21 DOH), and ceca (21 DOH) were linearly changed with the inclusion of sodium butyrate during posthatch feeding (Lan et al., 2020). Another study speculated that the relative weight of the duodenum peaked at 3 DOH, while there was a subsequent decline in relative intestinal growth through 21 DOH of heavy breed chickens (Dror et al., 1977). Similarly, duodenum villus area was greater in broiler vs. layer embryos at 14 DOI and continued through 7 DOH (Uni et al., 1995a,b). These authors also illustrated that the duodenum was the highest among other intestinal segments with greater enterocytes per villus in broiler embryo/chick, which increased with age. It can be concluded that the available bioactive substances, breed, and age of chicks affect GIT compartment development (BogusLAwska-Tryk et al., 2012).

Villus height (VH), crypt depth (CD), and the ratio between villus height and crypt depth (VH/CD) are the 3 most significant criteria that determine the developmental and functional state of the broiler GIT (Fan et al., 1997; Yamauchi, 2002; Xu et al., 2003; BogusLAwska-Tryk et al., 2012; Hassan et al., 2018; Reicher et al., 2022). The intestinal epithelium that covers the villi are invaginates into the lamina propria, forming tubular glands called intestinal crypts (Sobolewska et al., 2017b). In ovo feeding of methionine increase the VH, width, area, and height of enterocytes, which play a key role in nutrient absorption from the intestinal lumen into blood vessels (Potten and Loeffler, 1987), and goblet cell density (Nazem et al., 2017), arginine (Arg) increases the VH and lowers the CD in the duodenum (Gao et al., 2017c), and a 10% degraded date pits diet increases the VH and VH/CD, and lowers the CD of the broiler intestine (Alyileili et al., 2020). An increase in any of these morphometric parameters is anticipated to improve hydrolysis, immune system or barrier function and nutrient absorption (Awad et al., 2008; Salvi and Cowles, 2021), and the capabilities of the brush border membrane (Yamauchi, 2002). Deeper CD stimulates the secretion of digestive enzymes (Xu et al., 2003), and the formation of intestinal epithelial cells since it comprises of populations of continuously proliferating stem cells (Potten and Loeffler, 1987).

These ameliorative effects of the bioactive substances are primarily enhanced through the modulation of intestinal microflora composition (Bednarczyk et al., 2011, 2016; Siwek et al., 2018). Thus, GIT morphology, development, and physiology could be modulated through in ovo feeding approaches for the establishment of chicken posthatch homeostasis (Lilburn and Loeffle, 2015; Bogucka et al., 2017; Pender et al., 2017).

INTESTINAL MUCOSAL IMMUNITY

Intestinal mucosal immunity is the first barrier against pathogens (Muller et al., 2005). This immunity is achieved by highly efficient mucosal barrier and specialized multifaceted immune system; made up of a large population of scattered immune cells and the gut-associated lymphoid tissue (GALT) (Ahluwalia et al., 2017). The intestinal mucosa has more than 70% of immune cells (B cells, T cells, and macrophages), responsible for maintaining and control the GIT health of chickens (Muller et al., 2005). A well-developed GIT maintains immune homeostasis in chickens (Sobolewska et al., 2011; Bogucka et al., 2017; Sobolewska et al., 2017a).

The mucosal immune defense of the gut can be divided into 3 different anatomical parts; the intestinal epithelial barrier, the lamina propria, and the GALT. The GALT is the largest lymphatic organ of the body and is composed of 3 different entities of organized lymphoid tissues namely; Peyer's patches (PP), isolated lymphoid follicles (ILF), and mesenteric lymph nodes (MLN) (Mason et al., 2008). Mucosal immune defense can also be divided into inductive and effector sites. The inductive sites, where antigens sampled from mucosal surfaces activate naive and memory T and B lymphocytes consist of organized nodes of lymphoid follicles and include PP, ILF, and MLN (Brandtzaeg et al., 2008; Mason et al., 2008). The effector sites, where the effector cells after extravasation, retention, and differentiation perform their action, consist of the epithelium and the lamina propria where the lymphocytes are scattered throughout the tissue (Mowat, 2003; Brandtzaeg et al., 2008; Mason et al., 2008) (Figure 1).

Figure 1.

Immunity system modulation mechanism and antigen entrance routes. Innate immunity constitutes the first line of defense and is mediated by innate immune cells such as tissue macrophages, dendritic cells (DC), and granulocytes which elicit their effector function within minutes to hours following antigen exposure. Innate cells become activated via germ-line encoded pattern-recognition receptors (PRR), including toll-like receptors (TLR) and NOD-like receptors (NLRP) which recognize invariant features of pathogens (pathogen-associate molecular patterns or PAMPS) and tissue damage. Once activated, innate cells such as macrophages and neutrophils can effectively clear antigens via phagocytosis. Other types of innate cells, such as DC take up and process antigens, resulting in expression of antigenic epitopes. These DC can then serve as antigen-presenting cells (APC) for the priming of the adaptive immune system. In this way, the early innate response is coupled, and facilitates adaptive immunity. Antigens can enter through the microfold (M) cells in the follicle-associated epithelium (FAE) and be passed on to the dendritic cells (DCs), and the DCs then present the antigen directly to T-cells in the Peyer's patches or alternatively may reach the mesenteric lymph nodes (MLNs) through the draining lymph and subsequently be presented to naïve T-cells in the MLN. Another route involves direct antigen sampling of the intestinal lumen by DCs which extend dendrites between the epithelial cells to reach the lumen. Additionally, the antigens may gain entrance through the FAE enterocytes which can either pass antigens on to DCs or possibly act as local APCs via expression of MHC class II. 1. Activated antigen-specific clones, 2. Responding T lymphocyte, 3. Regulatory T lymphocyte, 4. Microbial antigen presented by antigen-presenting cell, 5. Infected cell expressing microbial antigen.

Embryonic feeding of bioactive substances has been proven to improve the immune response, and accelerate the development and maturation of the immune system of chickens (Dibner et al., 1998; Kadam et al., 2013; Murate et al., 2015; Qamar et al., 2020). This immune system modulation occurs directly through the interaction with immune cell receptors, stimulation of endocytosis, phagocytosis, and the production of numerous cytokines and chemokines (Schley and Field, 2002) as described in Figure 1. The status of the immune response has been recognized with markers including some cytokines, such as IL-1β, IL-10, IL-4, IL-6, IL-8, IL-18, IL-12P40, IFN-β, and IFN-γ (proinflammatory), and IL-1β and IL-10 (anti-inflammatory) (Tao and Elad, 2018; Wassie et al., 2021). In addition, the expression of CD3, CD45, CD56, chB6, CD80 (gene-associated immunity), TLR2, and TLR4 (toll-like receptors) are frequently used indicators of the immune response (Tao and Elad, 2018; Wassie et al., 2021).

Immune Cell Receptors

The host detects pathogen-associated molecular patterns (PAMPs) using innate immune sensors known as pattern-recognition receptors (PRRs), which mediate antimicrobial responses. PRRs are expressed by dendritic cells (DCs) and other phagocytic cells of the immune system and enable detection of microbes (Trinchieri and Sher, 2007; Peterson and Artis, 2014). Furthermore, PRRs are expressed by IECs on their surface as well as within their cytoplasm (Backhed and Hornef, 2003; Hurley and McCormick, 2004; Yuan et al., 2004; Trinchieri and Sher, 2007). PRRs are not only limited to TLRs, but can also bind to microbial compounds of both pathogenic and commensal bacteria. However, the differentiation mechanisms of PRRs in pathogenic and commensal organisms have not yet been fully elucidated (Mowat and Viney, 1997; Peterson and Artis, 2014).

TLRs exhibit various mechanisms action to prevent infection (Zhang and Liang, 2016), including regulating intestinal epithelial barrier integrity and modulating signaling pathways (Taghavi et al., 2008; Nighot et al., 2017; Rehman et al., 2021), regulating the expression of pro- and anti-inflammatory cytokines and avian beta-defensin (AvBD) (St Paul et al., 2013; Yoshimura, 2015; Kang et al., 2019; Shimizu et al., 2020; Terada et al., 2020), and playing pivotal roles in the initiation of innate immunity (Kawai and Akira, 2010; Rehman et al., 2021; Sarfraz et al., 2022).

The activation of TLRs is promoted by different bioactive substances in chickens (Sato et al., 2009; Terada et al., 2020; Rehman et al., 2021). Lactobacillus reuteri (LR) and Clostridium butyricum (CB) affect the innate immune system in broilers by modulating the expression of TLRs (Terada et al., 2020).

Ying et al. (2022) reported that the mRNA expression of TLR1A, TLR1B, and TLR2A was significantly downregulated in the ileum through dietary quercetin supplementation of Arbor Acre (AA) in broilers. These authors also reported that quercetin supplementation significantly downregulated the mRNA expression of MyD88, TIRAP/MAL, TBK1, IKK, NF-B, and IRF7 (Ying et al., 2022). Likewise, TLR2 and TLR4 mRNA expression was significantly higher after treatment with mannan oligosaccharides (Cheled-Shoval et al., 2011). In contrary, another study found that no significant difference was observed in the expression levels of TLR4, following raffinose injection in broilers (Berrocoso et al., 2017).

Activation of Lymphocytes and Phagocytosis

The intestinal lamina propria contains abundant B lymphocytes, especially IgA+ cells (Yang et al., 2021). These IgA+ cells form an important mucosal protective layer on the surface of the intestinal mucosa and play an important role in protecting the intestinal tract from pathogenic infection. In ovo injection of 1 and 2 mg/egg Astragalus polysaccharide (APS) at 18 DOI increased the IgA+ cells and improved sIgA content in the intestinal mucosa (Yang et al., 2021). Probiotics also influence humoral and cell-mediated immune responses by upregulating T lymphocyte numbers and associated responses (Brisbin et al., 2010; Lee et al., 2010). Similarly, the in ovo delivery of synbiotics has a modulation impact on the posthatching development of GALT, high colonization of GALT by T cells in the cecum, and enhanced B-cell proliferation in peripheral lymphatic organs (Siwek et al., 2018). Madej and Bednarczyk (2016) showed that in ovo feeding of prebiotics and synbiotics (inulin, transgalacto-oligosaccharides, Lactococcus lactis subsp. lactis IBB SL1 or Lactococcus lactis subsp. cremoris IBB SC1) impacted the composition of T cells and B cells in GALT: this increased diffuse lymphohistiocytic infiltration and solitary lymphoid follicles in the mucosa indicated an increased immunological response (Junaid et al., 2018). Consistently, dietary supplementation with Lactobacillus-based probiotics modulated the intraepithelial lymphocyte population that expresses the surface marker cluster of differentiation 4 (CD4) for resulting in induced intestinal immunity against coccidiosis (Zulkifli et al., 2000; Dalloul et al., 2003).

Augmenting the phagocytic capacity of macrophages, heterophil oxidative bursts, and degranulation are mechanisms action of bioactive compounds to enhance the innate immune system of chickens (Farnell et al., 2006; Higgins et al., 2007; Stringfellow et al., 2011; Pan and Yu, 2014). For instance, in ovo injection of prebiotics (0.76 mg/egg inulin +0.528 mg/egg Bi² tos) and synbiotics (0.76 mg/egg inulin +0.528 mg/egg Bi² tos + L. lactis subsp. cremoris IBB with 3 × 10⁸ living cells) on the 12th DOI enhanced a transient increase in the rate of Phag+ cells at 21 DOH (Stefaniak et al., 2019). Consistently, Higgins et al. (2007) demonstrated that administering a multistrain Lactobacillus probiotic (3 Lactobacillus bulgaricus, 3 Lactobacillus fermentum, 2 Lactobacillus casei, 2 Lactobacillus cellobiosus, and 1 Lactobacillus helveticus) in Salmonella enteritis exposed broilers reduced the number of macrophages in the ileum and ceca. The macrophage count reduction in infected birds attributed to a decrease in the bacterial load due to competitive exclusion mechanisms.

In Ovo Administered Bioactive Substances for the Production of Cytokines and Chemokines

Cytokine secretion by immune cells has been reported to stimulate GC proliferation and mucus production. For example, the secretion of IFN-g by the activation of the Th1 pathway, IL-13 by dendritic cells and macrophages, and IL-4, IL-5, IL-9, and IL-13 by T helper 2 cells have been used to stimulate GC proliferation and mucus production (Birchenough et al., 2015). The mucins that are primary secreted by goblet cells (GCs) are used to create a protective mucus layer as shown in Figure 2. Other GC proteins including IgA, lysozyme, and avidin also play major roles in the innate immunity of chickens (Bar Shira and Friedman, 2018). Regulated secretion of mucus is a rapid response to external stimuli as the first defensive mechanism of the gut. Glycosylation of O-glycan regulates the distribution of mucin types in GCs, which can be affected by both host and external factors, including pre/probiotic nutrients in the diet, inflammatory markers, hormones and neurotransmitters, and commensal and pathogenic bacteria (Duangnumsawang et al., 2021). The expression of immune-related genes can be either up- or downregulated.

Figure 2.

Probiotics reduce colonization of pathogens through competitive exclusion and enhance immune response; in ovo provided probiotics increase T lymphocyte numbers and modulate production of several proinflammatory includes T helper Type-1 (Th1)-dependent delayed-type hypersensitivity (DTH), IgG antibodies, T helper Type-2 (Th2) (IgE antibodies), T helper-17 (T17) cytokines; thus regulates the process and the development of regulatory T (Treg) cells in mesenteric lymph nodes; mucosal dendritic cells carry antigens to them and become conditioned for induction of T cells.

The immunomodulatory effects of in ovo administered bioactive substances in broiler were expressed with different modulation actions (Table 2). For instance, increasing antimicrobial peptide production (Farnell et al., 2006; Higgins et al., 2007; Stringfellow et al., 2011; Pan and Yu, 2014); can alter humoral and cell-mediated immune responses (Brisbin et al., 2010; Lee et al., 2010). Prebiotic B²Tos administration on 12 DOI upregulates IL-1β, IL-10, and IL 12p40 cytokines (Slawinska et al., 2019); synbiotics (1.9 mg raffinose with 3 × 10⁸ cfu Lactobacillus lactis subsp. lactis IBB) increased IL-4, IL-6, IL-18 and IFN-β in the spleen of chickens (Sławinska et al., 2014). However, synbiotics (1.76 mg inulin with 1,000 cfu of Lactobacillus lactis subsp. lactis 2955) and prebiotics (1.76 mg inulin) downregulates IFN-γ, IFN-β, IL18, IL-6, IL-4, IL-12p40, IL-8, and CD80 during the posthatch period (Płowiec et al., 2015). Likewise, in ovo administration of inulin or inulin supplemented with L. lactis subsp lactis 2955 on 12 DOI resulted in downregulation of immune-related genes in the spleen and cecal tonsils of broilers during the 35 d after hatching (Cheled-Shoval et al., 2011). More specifically, the expression levels of CD3 and chB6 in the small intestine of broilers were significantly upregulated by raffinose administration (Berrocoso et al., 2017). However, these authors confirmed that no significant difference was observed in the expression levels of CD56, TLR4, IL-1β, and IL-10 postraffinose injection in broilers. Madej and Bednarczyk (2016) also showed that in ovo feeding of prebiotics and synbiotics (inulin, transgalacto-oligosaccharides, Lactococcus lactis subsp. lactis IBB SL1 or Lactococcus lactis subsp. cremoris IBB SC1) had effects on the composition of T cells and B cells in GALT. Similar authors found that the number of CD3-expressing cells was increased by some synbiotics; however, there was no significant difference in the population of CD3- or chB6-expressing cells in only prebiotic-treated birds (Madej and Bednarczyk, 2016).

Table 2.

Effects of in ovo administered bioactive substances on the immune system of broilers.

Bioactive substances (pre, pro, and synbiotic)	Level or doze	Injection time	Site of injection	Effects or function	References
PrimaLac (Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium, and Bifidobacterium bifidum)	1 × 106 or 1 × 107 cfu	18 DOI	Air cell	Increase MUC2 expression	Pender et al., 2017
GOS (trade name: Bi2tos)	0.2 mL	12 DOI	Air cell	Increase IL-1β, IL-10, IL 12p40 expression	Slawinska et al., 2019
Raffinose with Lactobacillus lactis subsp. lactis IBB SL1	1.9 mg with 3 × 108 cfu, respectively	12 DOI	Air cell	Increase IL-4, IL-6, IL-18 and IFN-β expression	Sławinska et al., 2014
Inulin with Lactobacillus lactis subsp. lactis 2955	1.76 mg with 1,000 cfu	12 DOI	Air cell	Downregulates IFN-γ, IFN-β, IL18, IL-6, IL-4, IL-12p40, IL-8, and CD80 expression	Płowiec et al., 2015
Inulin	1.76 mg	12 DOI	Air cell	Downregulates IFN-γ, IFN-β, IL18, IL-6, IL-4, IL-12p40, IL-8, and CD80	Płowiec et al., 2015
B. subtilis, Pediococcus acidilactici, E. faecium	0.5 mL/egg with 107 CFU	18 DOI	Amniotic fluid	Increase ileal MUC2 gene expression in the late embryonic period	Majidi-Mosleh et al., 2017
Iinulin, Bi2tos 1.76 mg inulin + 1,000 CFU L. lactis subsp. Lactis IBB 0.528 mg of Bi2tos + 1,000 CFU of L. lactis subsp. cremoris IBB	1.76 mg of inulin 0.528 mg of Bi2tos 1.76 mg inulin + 1,000 CFU L. lactis subsp. Lactis IBB 0.528 mg of Bi2tos + 1,000 CFU of L. lactis subsp. cremoris IBB	12 DOI	Air cell	Modulate the production, maturation, and reactivity of leukocytes	Stefaniak et al., 2019
Toll-like receptor (TLR) 7 ligand/ Resiquimod	100 µg	18 DOI	Amniotic fluid	Upregulates the mRNA expression of IFN-γ and IL-1β in the lungs of chick	Senapathi et al., 2020

GOS, galacto-oligosaccharides.

GIT MICROBIOTA MODULATION THROUGH IN OVO BIOACTIVE SUBSTANCE DELIVERY

The GIT of chickens has diverse microenvironment with a complex population of microorganisms (Rolhion and Chassaing, 2016; Glendinning et al., 2019; Wang et al., 2023), and contributes to the host's gut homeostasis, immune status, and metabolism (Dunislawska et al., 2021). Inclusively, the intestinal microbiota plays pivotal role in nutrient digestion, growth performance, pathogen exclusion and the overall health of chickens (Stanley et al., 2014; Schokker et al., 2015; Sohail et al., 2015; Yadav and Jha, 2019; Wang et al., 2023). These improvements are linked with the modification of intestinal microbial communities (Angelakis and Raoult, 2010; Torok et al., 2011; Singh et al., 2012; De-Maesschalck et al., 2015; Pender et al., 2017; Wang et al., 2023). One promising option for microbial profile manipulation is the delivery of bioactive substances during the incubation stage of chicks (Gibson and Fuller, 2000; Doyle, 2001; Khan et al., 2014; Pender et al., 2017; Amrit et al., 2021; Ayalew et al., 2022). These microbial manipulations may change the bacterial composition, influence its developmental process, enhance the immune system, and maintain GIT integrity as well as the overall health of chickens, as shown in Figure 2 (Indikova et al., 2015; Pender et al., 2017; Rajesh et al., 2020; Abd El-Hack et al., 2021; Celina and Tamiris, 2021).

The population and structure of microbial communities are dynamic and could be affected by age (especially at the early stages of life), sex, diet or feed additives, phytobiotics, bacteriophages, and noninfectious and/infectious stressors (Clavijo and Florez, 2018; Diaz Carrasco et al., 2019). Furthermore, microbial community varied among intestinal segments (from crop to cloaca) and sampling sites (mucosal vs. luminal content). For instance, crop vs. ceca (the most predominant niche) had 10³ to 10⁴ CFU/g; Lactobacilli and Streptococci and 10¹¹ to 10¹² CFU/g; Ruminococci, Bacteroides, Clostridia, Streptococci, Enterococci, Lactobacilli, and E. coli, respectively (Yadav and Jha, 2019). In addition, the GIT microbiota reaches a mature state in between wk 2 and 3 posthatching (Huang et al., 2018); this is very late compared with the exposure time of environmental infectious threats staring on the first day of hatching. Consequently, this dysbiosis can disrupt the intestinal morphology and activities of chickens (Shang et al., 2018). Thus, maintaining and controlling natural microbial homeostasis in the GIT is crucial.

The intestinal microbiota was modulated through in ovo feeding of embryos. In ovo administration of wheat based prebiotic at17 DOI increased the intestinal Lactobacilli and Bifidobacteria populations (Tako et al., 2014). Another study demonstrated that in ovo injection of L. plantarum IBB3036 + lupin RFO-10⁵ CFU + 2 mg prebiotic, and L. salivarius IBB3154 + Bi²tos-10⁴ CFU probiotic + 2 mg at air chamber at18 DOI modulated GIT microbiota due to its adherence ability (Aleksandrzak-Piekarczyk et al., 2019). Furthermore, in ovo injection of L. acidophilus at a dose of 1 × 10⁶ at the amnion on 18 DOI significantly increased the concentration of probiotic bacteria Lactobacillus spp. and lowered the concentration of harmful microbes in the jejunal contents of broilers (Kanagaraju et al., 2019). Likewise, dietary MOS (1 g/kg) increases the Lactobacillus and Bifidobacterium content of the chicks intestine (Baurhoo et al., 2007), injection of 2 Lactobacillus strains, 1 Bifidobacterium strain, 1 Enterococcus strain, and 1 Pediococcus strain (a multibacterial species probiotic) modulates the composition and activities of the cecal microflora of broilers (Mountzouris et al., 2007), and Enteromorpha polysaccharide (EP) regulates the intestinal microbiota in chickens (Wassie et al., 2021). Therefore, in ovo delivery of bioactive substances has proven similar to dietary supplementation in terms of promoting a healthy microbial balance, and enhancing host defenses against several pathogens at the early stage of chicken development.

IN OVO BIOACTIVE SUBSTANCES EFFICACY FOR COMBATING PATHOGEN

The gut microbiota is one of the main defense components in the digestive tract against enteric pathogens. The disturbance of the gut microbiota–host interaction plays a crucial role in the development of intestinal disorders. For instance, cecal microbiota have been significantly changed in chickens infected with C. perfringens or Escherichia coli (Feng et al., 2010; Stanley et al., 2012; Skraban et al., 2013), Eimeria species (Perez et al., 2011; Stanley et al., 2014; Wu et al., 2014); and Salmonella Enteritidis (Nordentoft et al., 2011; Juricova et al., 2013; Videnska et al., 2013). With respect to maintaining the health of GIT microbiotas, several investigations of in ovo administered bioactive substances have been indicated (Table 3); these substances exert preventive and protective measures against different infection in chickens through various mechanisms of action. Thus, different bioactive substances could be used as a biological alternatives in combating chicken diseases by maintaining GIT microbial homeostasis as shown in Figure 2 (Holzapfel and Schillinger, 2002; Patterson and Burkholder, 2003; Siragusa and Ricke, 2012).

Table 3.

In ovo administration of bioactive substances for broilers against infection.

CONCLUSIONS AND IMPLICATIONS
Bioactive substance	Doze/level	Administration time	Function/infection recovery	References
PrimaLac (Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium, and Bifidobacterium bifidum)	1 × 106 or 1 × 107 cfu/egg	18 DOI	Reduce the severity of Coccidia	Pender et al., 2016
E. faecium M74 + Marek's vaccine (Merial)	1.4 × 107 CFU/egg	18 DOI	Improve Enterococci recovery	Skjøt Rasmussen, 2019
Methanolic root-bark extract of Adansonia digitata	250 mg/mL extract	18 DOI	Antiviral activity against Newcastle disease virus	Sulaiman et al., 2011
2 Bacillus amyloliquefaciens and 1 Bacillus subtilis	5 × 107 CFU/mL	18 DOI	Reduce the severity of virulent E. coli	Arreguin-Nava et al., 2019
GOS	3.5 mg/embryo	12 DOI	Reduce the incidence of intestinal lesions and oocyst excretion in chickens	Angwech et al., 2019
GOS	3.5 mg/embryo	12 DOI	Reduce foot pad dermatitis score	Slawinska et al., 2020
Arctostaphylos uvaursi extract	0.01 g	10 DOI	Modulate AFB1-induced toxicity in chicken embryos	Elwan et al., 2022
Synbiotic (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, and mannan oligosaccharide)	0.2 mL or 0.1% MOS, L. delbrueckii subsp. bulgaricus (103 cfu/egg) and S. salivarius subsp. thermophilus (103 cfu/egg).	18 DOI	Have positive effects on IBD antibody titers	Babazadeh and Asasi, 2021.

This table shows the probiotics species, dose level, and injection days used for the prevention or alleviation of enteric infections through in ovo techniques; they have different mechanisms including competitive exclusion, production of inhibitory substances, immune system modulation, and improved barrier function). CFU, colony-forming units; AFB1, aflatoxin B1; DOI, days of incubation.

Probiotics have been used for eliminating many economically important poultry diseases and pathogens (Dalloul and Lillehoj, 2005; Knap et al., 2010; Pender et al., 2016). Many strains of probiotic bacteria such as Enterococci, Bacilli, lactic acid bacteria, and yeast have used as anti-C. perfringens activities (Rajput et al., 2020). However, Yamawaki et al. (2013) and De Oliveira et al. (2014) demonstrated that probiotic (Lactobacillus spp.) injection into air cells or into the amniotic fluid on the 18th DOI did not protect against Salmonella Enteritidis challenge in cecum at 2 to 3 d of posthatch chicks. These inconsistent results may be attributed to the probiotic strain used, volume, delivery site, genotype, injection procedure, and hygiene practice variation. Although probiotics have different mechanisms of action, the mediation of mucosal immunity against infection is mainly mediated by the action of immunoglobulin A (sIgA), which can block the connection between pathogens and the epithelium; and cause bacterial agglutination (Mantis et al., 2011). Thus, intestinal sIgA levels can increase with supplementation of live yeast S. cerevisiae or S. boulardii in broiler chickens (Gao et al., 2009; Rajput et al., 2013). However, Wang et al. (2017) indicated that the supplementation of Kluyveromyces marxianus did not significantly influence the jejunal and ileal sIgA content, which confirmed the different physiological roles of Kluyveromyces marxianus compared with other yeast probiotics.

Similarly, in ovo or dietary supplementation of perbiotics (polysaccharides) including yeast beta-glucans enhanced gut health in chickens (Anwar et al., 2017), alleviated aflatoxin B (1)-induced DNA damage in lymphocytes (Zimmermann et al., 2015), and prevented C. perfringens-induced necrotic enteritis (Tian et al., 2016). Likewise, supplementation of mannan-oligosaccharides (MOS) in broilers increased Lactobacillus community diversity and decreased C. perfringens and E. coli in the ileum (Kim et al., 2011). Exploitation of the synergistic effect of different bioactive compounds with different molecular weights and compositions has gained increasing interest. This was evidenced in a study by Jen et al. (2021), where glycan with high molecular weights and polysaccharides composed of glucose (Glc), mannose, and galactose with low molecular weights exhibited synergistic effects on inhibiting proinflammatory mediator production. Similarly, the antiviral effects of combined polysaccharides from Eisenia arborea and Solieria filiformis exhibited the highest antiviral activities compared with the individual effects (Moran-Santibanez et al., 2016). Thus, the summary provided in this review indicates that the bioactivities of polysaccharides are closely linked with their structure and composition.

CONCLUSIONS AND IMPLICATIONS

In ovo administered prebiotics, probiotics, and synbiotics are helpful for GIT development, immune system function, and microbial homeostasis in chicks. The literature describes 2 major time points of in ovo delivery in chicken embryo development. The first time point is around 12 DOI, which is the main window for the delivery of prebiotics and synbiotics. The second time point is around 17/18 DOI, in which in ovo supplementation can mitigate the negative effects of starvation during the hatching window. However, functional ameliorative effects of bioactive substances have different in types and dosages, modes of action, sites of injection, and can have varying effects during various growing periods of chicks. Many studies have compared the beneficial effects of bioactive compounds at different doses and embryonic times of injection; however, no conclusive recommendation can be made under various confounding factors.

The mechanisms of action of bioactive substances are different, including antimicrobial peptide production, competitive exclusion, humoral and cell-mediated immune response alteration, phagocytic capacity, and degranulation. Mainly, these effects are helpful in the context of many economically important disease agents such as Eimeria, Newcastle disease virus, and infectious bursal disease virus. Although the benefits of bioactive substances are evident in numerous studies, further elucidation of their immunoregulatory effects on intestinal immunity under challenging conditions of disease is required. Moreover, further research will provide justification for biological compatibility of bioactive substances and hosts for the means of promoting early GIT development and improving the immune system, establishing beneficial bacteria, and enhancing gut health in chicks.