**3.1 Embryogenesis**

The avian egg contains both physical and chemical defense mechanisms to inhibit microbial invasion and proliferation. The eggshell has four physical defense mechanisms: (1) the cuticle, (2) the shell, (3) inner shell membrane, and (4) outer shell membrane [40]. Chemical defenses within the developing embryo include antimicrobial properties of the albumen, alkaline pH, lysozyme, and conalbumin/ovotransferrin [40]. Potential contamination of the egg occurs both before oviposition (trans-ovarian route) or after oviposition (trans-shell route; [41]). Environmental temperature and humidity are also known to impact the rate of microbial penetration of eggshells [42]. High relative humidity is considered essential for trans-shell transmission of microbes because it promotes survival, growth and transport through eggshell pores [43]. As the egg cools after lay, a relative vacuum is generated and the negative pressure facilitates microbial penetration of the eggshell [41]. Additionally, the quality and thickness of the eggshell impact a microbe's ability to penetrate the eggshell [44]. Comprehensive reviews describing microbial contamination of the egg and penetration of the eggshell have been published [5, 40, 41].

The composition of the neonate's GIT microflora is thought to be predominantly influenced by fecal and environmental contaminants on the eggshell [45], but the composition may also be affected by microbes vertically transmitted from hen to offspring at oviposition. Demonstrated that the hen's gastrointestinal tract microbiota influenced the composition of the chick's gut microbiota at hatch and there was a shared core microbial profile between the hen, embryo, and chick. There is further evidence of a partial transfer of the maternal oviduct microbiota to the embryo (progeny) during egg formation [46]. However, introduction of environmentallyderived microbial contaminants may complicate findings when using DNA sequencing to assess microbial profiles in samples, especially when sample number is low. Nevertheless, pathogen transmission during the perinatal period, either maternal, fecal, or environmentally-derived, leads to potential horizontal transmission of pathogens at the hatchery level. If contaminated hatching eggs are not sanitized properly before incubation, these eggs serve as a primary source of contamination in commercial hatcheries [2, 6, 7]. Both culture-based methods and sequencing techniques (culture-independent methods) have been applied to evaluate microbial presence on the surface of the eggshell. Using conventional microbiological techniques or culturebased methods, it was determined that eggshell surface contained ~1 *×* 103 colony forming units (CFU) per egg [47]. The composition of the eggshell microbiota of hatching eggs can be altered by the breeder hen's fecal microbiota or the environment. Buhr et al. [48] demonstrated that eggshell contamination negatively affected hatchability and surface sanitation of dirty eggs only marginally improved hatchability compared to non-sanitized dirty eggs. The eggshells of sanitized hatching eggs have been shown to harbor extensive numbers of microbes [49]. Additionally, sanitization of both clean and dirty hatching eggs increased total aerobic bacterial recovery from eggshells at the time of transfer (day 18 of embryogenesis) from incubator to hatch cabinet. However, nest-clean eggs that were not sanitized had lower total aerobic

#### *Value and Limitations of Formaldehyde for Hatch Cabinet Applications: The Search… DOI: http://dx.doi.org/10.5772/intechopen.104826*

bacterial recovery at transfer compared to the time of collection. Handling after the sanitization process should be limited to prevent contamination or recontamination of the surface of the eggshell. Potential for eggshell surface contamination occurs during egg collection, transport, artificial incubation, and hatching. It is important to limit the risk of contamination at each point throughout the egg collection and artificial hatching process.

Although there are physical and chemical defense mechanisms to prohibit microbial penetration of the eggshell and endogenous replication during embryogenesis, certain microbes have developed the ability to more readily penetrate the eggshell and evade host defenses. Certain Gram-negative bacteria, such as *Salmonella* can replicate on the eggshell surface at suboptimal temperature for growth and without supplemental nutrients [50]. At the time of lay, the eggshell may become contaminated with *Salmonella* by brief contact with contaminated nest box shavings [51]. Contamination of the eggshell surface with fecal material, nest box shavings, or egg-derived debris increased cultivable aerobic bacteria compared to clean eggs [52]. Using 16S RNA amplicon sequencing, Olsen et al. [52] showed that the eggshell surface microbiome of non-sanitized, dirty eggs and clean eggs were different, but variability between samples within the same group complicated the results. The authors suggested that environmental contaminants present on the eggshell could have influenced the results [52]. Furthermore, the composition of the microbiome depends on the bacterial DNA present at the time of sampling and cannot be used as a standalone metric to detect viable microorganisms [53]. In another study, 16S sequencing was used to compare the breeder hen's fecal microbiota to the eggshell microbiome in two independent flocks [54]. Of the eggshells that were sampled, Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes contributed to 90% of the overall microbiota [54]. Transfer of potentially pathogenic bacteria and those associated with spoilage from breeder hens to the eggshell surface, included *Salmonella, Escherichia coli,* and *Staphylococcus* spp. [54]. Maki et al. [55] showed that source or exposure to only eggshell-derived microbes, environment-derived microbes, or to both eggshell and environmentderived microbes modulate the composition of intestinal tract microbiota and fecal microbiota post-hatch. The eggs that were only subjected to the environment-derived microbes were sterilized prior to incubation which could have negatively affected the eggshell cuticle integrity. Also, any maternal microbes transferred during oviposition or that penetrated the eggshell may have confounded the results. Regardless, results published by Maki et al. [55] do indicate that intestinal pioneer colonization of the GIT is readily affected by source of contamination during the neonatal period.

For decades, early exposure to probiotics or beneficial microbes has been used to inhibit colonization of pathogenic microbes by competitive exclusion [56–58]. In addition to competitive exclusion and performance benefits, beneficial bacteria may also have immunomodulatory effects on the host [35, 36, 59]. However, the site of probiotic administration (air cell, amnion, allantoic sac), probiotic strain, dose, volume, and day of administration during embryonic development, all impact colonization efficiency and chick hatchability [60]. Early application by *in ovo* injection at DOE18 promotes uptake of the material (vaccine, probiotic, etc.) by the chick during the pipping process [61]. Teague et al. [62] administered FloraMax-B11, a lactic acid bacteria (LAB)-based probiotic, into the amnion of embryonated broiler eggs at DOE18. *In ovo* application of the probiotic reduced *Salmonella* colonization, improved early performance, and had no impact on Marek's vaccine efficacy [62]. Thus, *in ovo* administration could be utilized to promote early colonization by beneficial microbes in domestic poultry neonates.

Migration and colonization by a non-pathogenic, bioluminescent *E. coli* was more efficient when administered by *in ovo* application at DOE18 into the amnion as compared to the air cell [63]. Additionally, there was an increase in spleen weight at hatch related to *in ovo* administration into the amnion [63]. The authors hypothesized this to be associated with an accelerated immune development compared to those that received *E. coli* via *in ovo* air cell injection [63]. An increase in the weight of immune organs, including the spleen, was observed with probiotic supplementation has been reported and was attributed to improved immune stimulation [64–66]. A direct correlation between immunocompetence and the weight of the spleen has been described [67]. Although probiotics have been shown to stimulate immune development [35, 36, 59] and suppress pathogen colonization or invasion when administered by *in ovo* application [36, 62], certain microbes may be detrimental to embryonic development due to the rapid proliferation and accumulation of lethal byproducts within the embryo. For instance, *in ovo* administration with *Bacillus subtilis* negatively affected hatchability [68]. The authors hypothesized that *B. subtilis* produced enzymatic and metabolic byproducts that were detrimental to embryo development and contributed to the high percentage of late dead embryos compared to *Lactobacillus acidophilus* and *Bifidobacterium animalis* [68]. Alternatively, *in ovo* administration of Norum TM, a mixed *Bacillus* spp. culture containing vegetative cells of two *Bacillus amyloliquefaciens* and one *B. subtilis* isolate at DOE18 did not affect hatchability, markedly reduced enteric Gram-negative bacterial colonization a day 3 and day 7 post-hatch, and significantly improved early performance compared to the non-treated challenged group [69]. *In ovo* administration of with *Bacillus* spp. may inhibit colonization of opportunistic pathogens without hindering livability and early chick performance. Future studies should be conducted with potential candidate organisms to confirm feasibility for perinatal application.

The effect of *in ovo* administration (amnion, DOE18) with apathogenic Enterobacteriaceae or LAB on the cecal microbiome and intestinal proteome in broiler chicks have been evaluated [18, 70]. In these studies, *in ovo* application of *Citrobacter* spp. or LAB differentially altered the cecal microbiome at DOH and potentially at 10 days-of-age [18], and antioxidant effects were upregulated and inflammation was reduced in the GIT of chicks that received the LAB at day 18 of embryogenesis [70]. Though, *in ovo* administration with one strain of *Citrobacter* spp.*,* but not both, increased oxidative stress and proinflammatory responses in the GIT at DOH [70]. Rodrigues et al. [17, 71] evaluated the effect of apathogenic Enterobacteriaceae or LAB on the ileal microbiome of 10-day-old broiler chickens. In contrast to LAB, pioneer colonization by Enterobacteriaceae postponed maturation of the ileal microbiome [17] and was associated with impaired intestinal immune function [71]. Taken together, these studies suggest the pioneer colonizers of the GIT influenced the composition of the intestinal microbiome and modulated the host's enteric inflammatory response.

#### **3.2 Postnatal or post-hatch period**

The GIT is rapidly colonized by microbes present in the environment shortly after hatch and readily established 72 h post-hatch [72]. The composition of the microbiota is impacted by the individual host and age of the host [73]. The route of exposure (oral vs. environmental) to LAB at hatch influenced rate of colonization by beneficial pioneer colonizers and subsequent composition of the intestinal microbiome in

#### *Value and Limitations of Formaldehyde for Hatch Cabinet Applications: The Search… DOI: http://dx.doi.org/10.5772/intechopen.104826*

broiler chickens [74]. However, Stanley et al. [75] documented significant interchicken variation in the composition of the cecal microbiome in broiler chickens perhaps associated with the lack of exposure to the maternal microbiota and sanitation procedures in commercial hatcheries [75]. To artificially mimic the transfer of maternal microbiota to progeny, the cecal microbiota was collected from 1, 3, 16, 28, or 42-week-old hens and orally administered at DOH to chicks followed by *Salmonella* Enteritidis challenge at day 2 [76]. Chicks that received cecal microbiota from 3, 16, 28, and 42-week-old of hens inhibited SE colonization in the ceca significantly compared to the non-treated, challenged control 4 days post-challenge [76]. However, administration of the cecal microbiota as a therapeutic treatment after oral challenge treatment with SE was not protective [76]. To investigate the rate of natural transfer of the maternal microbiota from hen to progeny, chicks were placed in contact with hens for 24 h post-hatch [77]. It was shown that exposure and transfer of the maternal microflora influenced the chick's cecal microbiota [77].

Administration of beneficial bacteria has been shown to inhibit pathogen colonization and reduce horizontal transmission of pathogenic bacteria [78, 79]. Early establishment of beneficial pioneer colonizers is critical for pathogen exclusion since the GIT is rapidly colonized the initial microbes in the environment at hatch. The pioneer colonizers of the GIT influence immune and metabolic functions that regulate host resistance to pathogens and tolerance of the commensal microbiota. Since commercially-reared poultry neonates do not have any contact with the hen at hatch, microbes present in fecal material or that predominate in the environment at the time of lay or hatch dictate the composition of the pioneer colonizers of the GIT. Artificial exposure to beneficial microbes during the perinatal period may improve poultry health and wellbeing in integrated poultry production systems where prophylactics and therapeutics are more limited than ever due to multi-drug resistance and shift towards antibiotic-free production.
