Commercially produced chickens have become key food-producing animals in the global food system. The scale of production in industrial settings has changed management systems to a point now very far from traditional methods. During the perinatal period, newly hatched chicks undergo processing, vaccination and transportation, which introduces a gap in access to feed and water. This gap, referred to as the hatching window, dampens the potential for microflora inoculation and as such, prevents proper microbiome, gastrointestinal system and innate immunity development. As a consequence, the industrial production of chickens with a poor microbial profile leads to enteric microbial infestation and infectious disease outbreaks, which became even more prevalent after the withdrawal of antibiotic growth promoters on many world markets (e.g., the EU). This review presents the rationale, methodology and life-long effects of in ovo stimulation of chicken microflora. In ovo stimulation provides efficient embryonic microbiome colonization with commensal microflora during the perinatal period. A carefully selected bioactive formulation (prebiotics, probiotics alone or combined into synbiotics) is delivered into the air cell of the egg on day 12 of egg incubation. The prebiotic penetrates the outer and inner egg membranes and stimulates development on the innate microflora in the embryonic guts. Probiotics are available after the mechanical breakage of the shell membranes by the chick’s beak at the beginning of hatching (day 19). The intestinal microflora after in ovo stimulation is potent enough for competitive exclusion and programs the lifespan condition. We present the effects of different combinations of prebiotic and probiotic delivered in ovo on day 12 of egg incubation on microflora, growth traits, feed efficiency, intestinal morphology, meat microstructure and quality, immune system development, physiological characteristics and the transcriptome of the broiler chickens. We discuss the differences between in ovo stimulation (day 12 of egg incubation) and in ovo feeding (days 17–18 of egg incubation) and speculate about possible future developments in this field. In summary, decades of research on in ovo stimulation and the lifelong effects support this method as efficient programming of lifespan conditions in commercially raised chickens.

Epigenetics is defined as the study of changes in gene function that are mitotically or meiotically heritable and do not lead to a change in DNA sequence. Epigenetic modifications are important mechanisms that fine tune the expression of genes in response to extracellular signals and environmental changes. In vertebrates, crucial epigenetic reprogramming events occur during early embryogenesis and germ cell development. Chicken embryo, which develops external to the mother's body, can be easily manipulated in vivo and in vitro, and hence, it is an excellent model for performing epigenetic studies. Environmental factors such as temperature can affect the development of an embryo into the phenotype of an adult. A better understanding of the environmental impact on embryo development can be achieved by analyzing the direct effects of epigenetic modifications as well as their molecular background and their intergenerational and transgenerational inheritance. In this overview, the current possibility of epigenetic changes during chicken embryonic development and their effects on long-term postembryonic development are discussed.

In ovo delivery of prebiotics and synbiotics in chickens allows for the development of intestinal microflora prior to hatching, which boosts their robustness. The goal of this study was to determine the transcriptomic profile of the spleen (S), cecal tonsils (CT), and large intestine (LI) of adult chickens injected with prebiotics and synbiotics in ovo. On day 12 of embryo development, incubating eggs were injected with prebiotics: inulin alone (P1) or in combination with Lactococcus lactis subsp. lactis IBB2955 (S1), galactooligosaccharides (GOS) alone (P2) or in combination with Lactococcus lactis subsp. cremoris IBB477 (S2); control group (C) was mock injected with physiological saline. Gene expression analysis was conducted using an Affymetrix Chicken Gene 1.1 ST Array Strip. Most of the differentially expressed genes (DEG) were detected in the cecal tonsils of P2 (378 DEG), and were assigned to gene ontology categories: lymphocyte proliferation, activation and differentiation, and cytokine production. Ingenuity pathway analysis of the DEG (CT of P2) indicated the inhibition of humoral and cellular immune responses, e.g., role of NFAT in regulation of immune responses, phagocytosis, production of nitric oxide, NF-κB, IL-8, and CXCR4 signaling. The DEG with the highest up-regulation from S1 and P2 were involved in gene expression (PAPOLA, RPL27A, RPLP1, and RPS29) from P1 and P2 in transport (BEST4, SLC9A3, and SLC13A2), metabolism (OGT, ALPP, CA4, and CA7), signaling (FGG, G3BP2, UBB, G3BP2, CACNA1G, and ATP6V0A4), and immune responses (MSMB, LGALS3, CABIN1, CXCR5, PAX5, and TNFRSF14). Two DEG influencing the complement system (SERPING1 and MIR1674) were down-regulated in P2 and S1. In conclusion, GOS injected in ovo provided the most potent stimulation of the host transcriptome. This is likely due to its strong bifidogenic effect, which triggers proliferation of indigenous embryonic microflora in ovo, and indirectly influences gene expression regulation in host tissues, especially cecal tonsils.

A previous study showed that prebiotics and synbiotics administered in ovo into the egg air cell on the 12th day of incubation enhance the growth and development of chickens. However, the influence of this procedure on the development and efficiency of the innate immune system of broiler chickens is unclear. Therefore, the aim of this study was to evaluate whether the early (on the 12th day of embryo development) in ovo administration of selected prebiotics (inulin − Pre1 and Bi2tos − Pre2) and synbiotics (inulin + Lactococcus lactis subsp. lactis IBB SL1 − Syn1 and Bi2tos + L. lactis subsp. cremoris IBB SC1 − Syn2) influences the innate immune system. Chickens (broiler, Ross 308) that were treated with Pre1 exhibited a decreased H/L ratio on D7, but an increased H/L ratio was observed on D21 and D35. In the remaining experimental groups, an increase in the H/L ratio was observed on D21 and D35. The oxidative potential of leukocytes measured using the NBT test increased on D21 in Pre2 and Syn1 groups. The rate of the phagocytic ability of leukocytes increased in Pre1 and Syn1 groups on D21. The phagocytic index decreased in Pre1 and Syn2 groups on D21 and D35. Concurrently, the count of WBC in circulating blood decreased on D21 in Pre1, Pre2, and Syn1 groups. The hematocrit value was increased in Syn1 chickens on D21, in Pre1 chickens on D35, and in Syn2 chickens on both time points. Early in ovo treatment of chicken embryos with prebiotics and synbiotics may temporarily modulate not only the production/maturation of leukocytes but also their reactivity.

Among various feed additives currently used in poultry nutrition, an important role is played by bioactive substances, including prebiotics. The beneficial effect of these bioactive substances on the gastrointestinal tract and immune system give rise to improvements in broiler health and performance nutrition, thus increasing the productivity of these birds. An innovative method for introducing bioactive substances into chickens is the in ovo injection into eggs intended for hatching. The aim of the study was to evaluate the development of histomorphological parameters of the duodenum and productivity in chickens injected in ovo with the prebiotic DiNovo® (extract of Laminaria species of seaweed, BioAtlantis Ltd., Ireland) on d 12 of incubation, under large - scale, high density poultry production conditions. There was no significant impact of the injection of DiNovo® prebiotic on the production parameters of broiler chickens (body weight, FCR, EBI and mortality) obtained on d 42 of rearing. No significant impact of the DiNovo® injection on the duodenum weight and length was observed, as well as on the CSA, diameter and muscular layer thickness of the duodenum. The in ovo injection of DiNovo® significantly increased the width of the duodenal villi (P < 0.05) and crypt depth (P < 0.01) of chickens on d 21 of rearing. Other histomorphological parameters of duodenal villi at d 42 of chickens rearing such as: the height, width, and cross section area of villi were significantly greater in chickens from the control group compared to those from the DiNovo® group (P < 0.05 and P < 0.01). In conclusion, this study demonstrates that injection of DiNovo® prebiotic into the air chamber of egg significantly influences the histomorphological parameters on d 21 of rearing without negatively affecting productivity in chickens at the end of rearing.