Pre-hatching and post-hatching environmental factors related to epigenetic mechanisms in poultry

Abstract

Epigenetic modifications are phenotypic changes unrelated to the modification of the DNA sequence. These modifications are essential for regulating cellular differentiation and organism development. In this case, epigenetics controls how the animal’s genetic potential is used. The main epigenetic mechanisms are microRNA activity, DNA methylation, and histone modification. The literature has repeatedly shown that environmental modulation has a significant influence on the regulation of epigenetic mechanisms in poultry. The aim of this review is to give an overview of the current state of the knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of the health status in chickens and other poultry species. One of the main differences between birds and mammals is the stage of embryonic development. The bird’s embryo develops outside its mother, so an optimal environment of egg incubation before hatching is crucial for development. It is also the moment when many factors influence the activation of epigenetic mechanisms, i.e., incubation temperature, humidity, light, as well as in ovo treatments. Epigenome of the adult birds might be modulated by nutrition, supplementation, and treatment, as well as modification of the intestinal microbiota. In addition, the activation of epigenetic mechanisms is influenced by pathogens (i.e., pathogenic bacteria, toxins, viruses, and fungi) as well as the maintenance conditions. Farm animal epigenetics is still a big challenge for scientists. This is a research area with many open questions. Modern methods of epigenetic analysis can serve both in the analysis of biological mechanisms and in the research and applied to production system, poultry health, and welfare.

Lay Summary

Epigenetic modifications are phenotypic changes unrelated to the modification of the DNA sequence. In this case, epigenetics controls how the animal’s genetic potential is used. The literature has shown that environmental modulation has a significant influence on the regulation of epigenetic mechanisms in poultry. The aim of this review is to give an overview of the current state of the knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of the health status in poultry. The bird’s embryo develops outside its mother, so an optimal environment of egg incubation before hatching is crucial for development. It is also the moment when many factors influence the activation of epigenetic mechanisms, i.e., incubation temperature, humidity, light, as well as in ovo treatments. Epigenome of the adult birds might be modulated by nutrition, supplementation, and treatment, as well as modification of the intestinal microbiota. The activation of epigenetic mechanisms is influenced by pathogens as well as the maintenance conditions. Farm animal epigenetics is still a big challenge for scientists. Modern methods of epigenetic analysis can serve both in the analysis of biological mechanisms and in the research and applied to production system, poultry health, and welfare.

Farm animal epigenetics is still a big challenge for scientists and it is a research area with many open questions. This review provides an overview of the state of the knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of the health status in chickens and other poultry species.

Introduction

Phenotypic background of each organism is coded in the DNA. Changes in the phenotype unrelated to the modification of the DNA sequence are called epigenetic modifications. Under the influence of many different environmental factors, such as nutrition, temperature, and stress (Figure 1), the multi-stage process of translation and transcription of genetic material to protein is changed. This change may result in a modulation of the phenotypic traits of individual (Cao et al., 2013). The most important epigenetic mechanisms include microRNA (miRNA) activity, DNA methylation, and histone modification. miRNAs are a fraction of small RNA molecules encoded in the genome that have a fundamental effect on gene expression. Mature miRNA binds to the 3’-UTRs end of the mRNA molecule of the regulated target gene, destabilizing it and preventing translation. In this way, the miRNA influences the silencing of target genes (Taganov et al., 2007). DNA methylation is the addition of methyl residues to cytosines within the CpG islands, which blocks enzymes from accessing the DNA. Hence, the transcription of genes from DNA into mRNA is inhibited (Shen and Waterland, 2007). DNA requires methyl donors and cofactors from the external environment. Therefore, the methylation process is influenced by many nutritional components and supplementation (Dunislawska et al., 2021b). In the case of histone modification, the enzymes responsible for this mechanism regulate transcription and are sensitive to the availability of endogenous small molecule metabolites. As a result, chromatin reacts to changes in the environment. For example, intestinal microbiota changes regulate methylation and acetylation of histones in host tissues in a diet-dependent manner (Stoll et al., 2018).

Figure 1.

Factors influencing epigenetic changes in poultry (created in BioRender.com).

The aim of this review is to give an overview of the current state of knowledge in poultry epigenetics in terms of issues relevant to overall poultry production and the improvement of health status in chickens and other poultry species.

Basics of Epigenetic Mechanisms

The DNA methylation is an epigenetic modification that can be gene specific or global. DNA methylation takes place at the cytosine residues of cytosine-phosphate-guanine dinucleotides (CpG) under the action of DNA methyltransferases (DNMT). Histone methylation, on the other hand, is catalyzed by histone methyltransferases (HMT). Histone methylation occurs at the N-terminal tail of the histone and depends on a specific amino acid residue, such as lysine or arginine. These modifications can affect gene expression depending on the particular amino acid that is methylated and the amount of these amino acids. Both DNMT and HMT require the methyl donor S-adenosylmethionine (SAM) to function (Mentch and Locasale, 2016). This indicates that the availability of SAM within one-carbon metabolism is of key importance in the regulation of gene expression and, consequently, in the proper functioning of the organism. The main source of methyl groups derived from SAM as a result of the methylation reaction is micronutrients supplied in the diet. These include B vitamins, especially B9 (folate), choline, methionine, and betaine (Niculescu and Zeisel, 2002). Choline is irreversibly oxidized by choline oxidase to betaine, providing one-carbon units in the conversion of homocysteine (Hcy) to methionine and generation of the universal methyl donor S-adenosylmethionine (SAM). After donating its methyl group, SAM is converted into S-adenosylhomocysteine (SAH), which is an inhibitor of methyltransferases. SAH is then hydrolyzed to homocysteine by S-adenosylhomocysteine hydrolase (SAHH). Homocysteine can be converted back to methionine by transfer of the methyl group from 5Mthf via methionine synthase, which requires cofactors vitamins B9 and B12 for its activity. Homocysteine can also be converted into cystathionine and then into cysteine (Bekdash, 2021).

Histone acetylation is a modulator of the chromatin structure that is involved in DNA replication and repair, gene transcription, and heterochromatin silencing. Histone deacetylase (HDAC) is an enzyme that removes the acetyl group from histone proteins on DNA. This makes DNA less accessible to transcription factors (Delcuve et al. 2012). Inhibition of HDAC activity may result in transcription reprogramming. HDAC regulates the activity and expression of numerous protein. Its inhibition affects the expression of about 20% of genes, without indicating a specific direction of regulation (Smith and Workman, 2009).

Another mechanism that may affect the silencing or alteration of gene expression is RNA interference. This is achieved by a double-stranded RNA with a structure and sequence similar to the sequence of the gene to be switched off. Switching off gene expression may occur by degrading or blocking mRNA translation, but also by inducing epigenetic gene silencing. RNA interference is mediated by small, double-stranded RNA. They are the product of nuclease processing of larger RNA fragments. Behind mRNA degradation are small interfering RNAs (siRNAs). They are characterized by complete sequence homology to the target sequence. As a consequence of degradation, the gene is silenced. Behind the process of interference with mRNA translation are microRNAs (miRNAs) (Svoboda, 2020).

Pre-hatch Effects in Chicken

Pre-hatching epigenetic modifications in chicken

One of the major differences between mammals and birds is that embryonic development of birds takes place outside the mother and thus an optimal prehatch environment is crucial for their development. According to Tzschentke and Plagemann, 2006, development of functional systems takes place during the perinatal period of organisms which is also known as a critical period. During this period, external environment can influence this functional systems development for the lifetime via neuroorganization or modification of expression of effector genes which is known as imprinting (Tzschentke and Plagemann, 2006). Prehatch environment of birds can be attributed to incubation conditions such as temperature, light, and humidity, composition of eggs, and other in ovo treatments. In chickens, the prenatal environment can be divided into the pre-lay and the incubation period. This means that the mother’s environment and the incubation period of the egg can have an impact to the offspring (Henriksen et al., 2011). Many phenotypic traits may be influenced by environmental factors through epigenetic mechanisms (Feil and Fraga, 2012). Factors influencing the early stages of life (prenatal and perinatal) are of particular importance for the development of the adult phenotype. The epigenome that develops during embryogenesis can persist during entire lifetime, reducing susceptibility to environmental changes (Skinner, 2011).

In vertebrates, crucial epigenetic reprogramming events occur during germ cell development and early embryogenesis. The precursors of reproductive cells are primordial germ cells (PGCs). Especially, the avian PGCs have a potential to be used as an important cell-based system for epigenetic study and in study on reproduction of vertebrates (Bednarczyk et al., 2021). PGCs can be transferred from one embryo to another and create functional germ cells in the embryo recipient (Sang, 1994; Chojnacka-Puchta et al., 2015). Molecular comparison of Green-legged Patridgelike PGCs with White Leghorn PGCs proved that the differences are determined by genetic and environmental factors. Genotype and gender have a significant influence on these epigenetic changes. PGCs were isolated from gonads from the two breeds during embryonic development. Global DNA methylation analysis showed significant changes in methylation levels in the gonadal PGCs of both breeds. The obtained results suggest a faster embryonic development of the Green-legged Patridgelike compared to the White leghorn embryos (Dunislawska et al., 2021b).

Chromatin remodeling due to events such as demethylation and remethylation of the embryonic genome following fertilization is a key step in epigenetic reprogramming (Wang et al., 2014). During the migration of primary germ cells to the gonads, they transform the obtained DNA methylation patterns and histone marks (Seki et al., 2005). Comparative studies on post-translational modifications in mammalian stem cells (mouse model) and chickens have shown that the nuclear distribution of these modifications is generally similar. However, there are some significant differences. Chicken embryonic stem cells (ECS) contain a high level of the trimethylated histone H3 on lysine 27 (H3K27me3), while PGCs contain high levels of the tri-methylated histone H3 on lysine 9 (H3K9me3) compared to mammalian stem cells. In addition, in chicken PGCs, increased H3K9me3 methylation was revealed, and gene expression involved in de novo DNA methylation is strongly expressed (Kress et al., 2016). This uniqueness of the primary germ cells of chickens may offer special opportunities to re-program the epigenome of the developing embryo by interfering with its environment at an early stage.

The specificity of the poultry production system in commercial hatcheries allows easy manipulation of the ambient temperature of the developing embryos. Pre-hatching exposure to higher temperature during the embryonic development of chickens between the 7th and 16th day of incubation may result in better thermotolerance in production-mature chicks without production losses (Piestun et al., 2008b). According to the authors, this period (7th–16th day of egg incubation) is crucial for the development of the hypothalamus-hypophysis-thyroid axis (thermoregulation) and the hypothalamus-hypophysis-adrenal axis (stress response) (Piestun et al., 2008a). As these studies did not fully explain the mechanisms by which better tolerance to heat stress was observed in production-mature broilers, researchers hypothesized that epigenetic phenomena were involved. David et al. (2019) provided the first evidence of an involvement of epigenetic mechanisms in the process of temperature conditioning during embryonic development. These authors showed that the introduction of a thermal conditioning protocol (39.5 °C for 12h/d between the 7th and the 16th day of embryogenesis) for chicken embryos influences post-translational modifications of the tri-methylation of lysine 4 on histone H3 (H3K4me3) and H3K27me3 histones in the hypothalamus and muscle tissues, known to contribute to environmental memory (the changes included genes related to neurodevelopmental, metabolic and regulatory functions). Data on changes in methylation patterns as a result of manipulation of incubation parameters (temperature, CO₂ concentration) of broiler eggs were provided by Corbett et al. (2020). They compared patterns of methylation in the heart tissue of chickens incubated during embryonic development at a standard temperature (37.8 °C) and temperature raised to 38.9 °C and at low (0.1%), medium (0.4%), and high (0.8%) CO₂ concentrations. The results obtained by reduced representation bisulfate sequencing (RRBS) showed thousands of loci with differential methylation associated with cardiac development in the compared groups. This confirms that manipulation of broiler egg incubation conditions may affect cardiac growth and physiology by altering DNA methylation patterns. We still have a very limited information related to the epigenetic influence of the incubation environment of chicken eggs. However, the ease of manipulating the parameters of the incubator provides scientists with the possibility of further intensive research on the epigenome of chickens under the influence of the incubator environment.

Maternal and transgenerational epigenetic effect

An interesting phenomenon is that the epigenetic modifications can be passed on from generation to generation as a result of transgenerational inheritance, despite the lack of exposure of subsequent generations to a given factor causing primary changes in the parental generation (Jablonka and Raz, 2009). Years of research have provided some evidence for intergenerational effects, but there is still little work on germline-dependent epigenetic mechanisms as the cause of non-DNA sequence inheritance in poultry (reviewed by Guerrero-Bosagna et al., 2018). Probably both chickens and mammals in the early stage of embryonic development have two significant moments (blastocyst implantation and migration of PGCs to the gonads) when epigenetic reprogramming occurs, during which some methylation patterns obtained from the parental generation may be lost (Reik et al., 2001; Seki et al., 2005).

It appears that research into transgenerational epigenetic inheritance in chickens may be difficult due to the non-rigid nature of most of the available chicken lines. In this case, it is difficult to distinguish between genetic and epigenetic effects (Leroux et al., 2017). However, the chicken model has some advantages in terms of the epigenetic transgenerational effect. Birds belong to oviparous species, and the development of the embryo, from a very early stage of embryogenesis, takes place outside the mother’s body. In the commercial production, the mother environment effect is limited to the composition of the egg. Moreover, of all livestock species, birds have the shortest replacement period, and the number of offspring is not limited to the available space of the mother’s uterus (Frésard et al., 2013).

Goerlich et al. (2012) show that chickens subjected to periodic social isolation for 3 weeks after hatching had a reduced response to limiting stress in later life; 29-d-old male offspring obtained from these animals also reacted with a suppressed corticosterone response to the same stress factor. Studies on the impact of maternal stress on subsequent generations assume that intergenerational effects of stress are probably transmitted by modulation of the hypothalamic-pituitary-adrenal axis, as well as by epigenetic mechanisms causing hereditary changes in gene expression. Stress in the early stages of life causes suppressed corticosterone reaction in experimental birds and consequently also in offspring. Stress-related genes, such as EGR1 and CRHR1, underwent positive regulation. The differences in the level of gene expression were also correlated with the generations (Goerlich et al., 2012). This allows the conclusion that, as in mammals (Roth et al., 2009), in birds there is a change in DNA methylation patterns that can be passed on through epigenetic transgenerational inheritance. However, more research is needed using molecular tools on this phenomenon in chickens.

Post-hatch Effects in Chicken

Epigenetic programming in chicken induced by in ovo delivered bioactive substances

In ovo feeding (IOF) offers great opportunities for an early embryo support, which can also be used to assess the effect of nutrients on epigenome changes in adult birds. Administering folic acid to the yolk sac on the 11th day of embryonic development of broiler chicken, induced methylation of histones in the promoters IL2 and IL4 showed enrichment in H3K4m2 and loss of K3K9me2 post-hatch in growing chicken (Li et al., 2016). In contrast, the IL6 promoter showed a decrease in K3K4m2 and an increase in K3K9me2. The obtained results suggest that folic acid administered with IOF has an impact on immune functions through an epigenetic regulation of immune genes.

In ovo administration of zinc on the embryonic development of zinc-deficient chicken eggs reduces embryo mortality and increases hatchability (Sun et al., 2018). In ovo administration of zinc compared with control non-injected group did not affect the overall DNA level of methylation and histone 3 lysine 9 (H3K9) acetylation of the MT4 promoter in chicken embryo liver. On the other hand, the administered organic zinc increased DNA methylation and acetylation as compared to inorganic zinc, which may indicate a higher efficiency of inorganic zinc in enhancing methylation and acetylation.

It has been shown that in ovo injection of betaine regulates cholesterol metabolism in the liver of chickens by activating epigenetic mechanisms (DNA and histone methylation) (Hu et al., 2015). Betaine affects the level of CpG methylation of the CYP7A1 genes (responsible for the conversion of cholesterol into gallstones) and ABCA1 (associated with cholesterol counter transport). These changes are associated with the level of expression of these genes. Betaine also alleviates effects of fatty liver in chickens related to diet and also effects associated with overexposure to corticosterone. In the experimental trail performed by Hu et al. (2017), betaine has been administered to fertilized eggs, while after hatching, 8-wk-old birds were administered subcutaneous corticostetone (Hu et al., 2017). It has been proven that the hepatic sweetening was less pronounced in chickens which received betaine in ovo. This effect was associated with increased expression of PPARα, CPT1α, and also mitochondrial DNA (mtDNA)-encoded genes. In addition, betaine influenced the activation of epigenetic mechanisms expressed by changes in the methionine cycle genes associated with CpG methylation modifications on the CPT1a and mtDNA D-loop regions gene promoter (Hu et al., 2017).

The liver, as the main metabolic organ, plays a key role in the metabolism of nutrients, digestion of fats, in the hormonal balance, and synthesis of blood proteins. It exhibits unique immunological properties due to the presence of the immune cell receptor (Racanelli and Rehermann, 2006; Szabo, 2015). Its anatomy enables close interaction with the intestines through the biliary tract, portal vein, and systemic circulation (the so-called enterohepatic axis) (Tripathi et al., 2018). Hence, the functioning of the liver has a significant impact on the intestinal bacteria and subsequently on the entire host organism. The peri-hatching period is crucial for programming the microbiota to enable colonization of the embryo’s intestines with beneficial bacteria before hatching. It has been shown that the administration of a single dose of prebiotic or synbiotic suspension to the egg’s air chamber on the day 12 of incubation provides effects that are visible through the entire chicken lifespan. Molecular changes are strongly manifested in the liver and spleen (Dunislawska et al., 2017; Siwek et al., 2018). To analyze the relationship between early stimulation of the intestinal microbiota and methylation in the chicken liver, on the day 12 of eggs incubation into the air chamber, synbiotics based on Lactobacillus strains were administered (Dunislawska et al., 2020). Analysis of DNA methylation in the liver showed that in ovo administration of the synbiotic Lactobacillus plantarum with raffinose family oligosaccharides leads to hypermethylation of the ANGPTL4 gene in liver, which is associated with silencing of its expression (10-fold decrease in expression after stimulation). High methylation is related to low or no transcription. The protein encoded by the ANGPTL4 gene is involved in lipid metabolism, insulin sensitivity, and glucose homeostasis.

Epigenetic regulation of gene expression by early stimulation of the intestinal microbiota is also dependent on miRNA activity, as demonstrated by analyzing this mechanism in the liver (Sikorska et al., 2021). The major methyltransferases in animals are believed to be regulated by miRNAs, so it can be important reasons for linking miRNA activity with modification of DNA methylation by interaction with newly formed miRNA strands of a given target gene (Chuang and Jones, 2007). Significant changes in miRNA activity in chicken liver after administration of probiotic and synbiotic in ovo have been demonstrated (Sikorska et al., 2021). It might be concluded that miRNA is an important element of the molecular mechanism of host-microbiota interaction, particularly in the context of gene expression silencing.

The analysis of the epigenetic mechanism of expression silencing of immune-related genes by in ovo stimulation was carried out on the spleen (as a main immune organ) of adult chickens after injection with probiotic, prebiotic, and synbiotic on day 12 of egg incubation. Moreover, the analysis covered two different genotypes—chicken broiler and Polish native breed. The analysis of the global methylation level in the spleen showed statistically significant differences between the two analyzed genotypes of chickens. They are also characterized by different levels of methylation of individual genes. In the case of the Polish native breed, no significant correlation was observed between the expression and methylation of genes, while this relationship was strongly noticeable in the chicken broiler (IKZF1, NR4A3, NFATC1 genes). The Ross 308 chicken broiler used in the experiment is a product line that was created as a result of an intensive genetic selection program. It is characterized primarily by a fast growth rate and excellent production parameters. The Polish native chicken (Green-legged patridgelike) is a breed of general use, which is characterized by high resistance to diseases, good laying characteristics, and low environmental and nutritional requirements. All these phenotypic differentiation characteristics may result in a differential response to the stimulation of the gut microbiota. It can be assumed that the given substances do not constitute a sufficiently strong environmental signal for the native breed of chickens, so that the epigenetic mechanism is not activated (Dunislawska et al., 2021b).

Chicken nutrition and host-microbiome interaction

The epigenome might be modulated by nutritional factors such as bioactives added to food (Figure 2), diet and its components, and also by seasons and times of the day, factors causing diseases: viruses, fungi, pathogenic bacteria, toxins, and antibiotics (Ognik et al., 2020). The methylation process can be also influenced by many diet components such as selenium, folic acid, flavonoids, or probiotic bacteria (Jaenisch and Bird, 2003). Changes in the level of methylation of DNA, leading to modification of gene expression, may also be affected by the prebiotic fermentation product—butyrate, a short-chain fatty acid (Pan et al., 2009; Paul et al., 2015). As described earlier, the nutrients like an in ovo stimulation cause changes in gene expression by affecting transcriptional and post-transcriptional mechanisms. They can also affect epigenetic mechanisms, causing long-term changes in gene expression (Waterland and Jirtle, 2004; Dunislawska et al., 2021a).

Figure 2.

Modification of the gut microbiota influences epigenetic changes (created in BioRender.com).

Maternal nutrition has a significant impact on the epigenome of the future offspring. This process is known as the maternal effect. It is a non-genetic interference by the mother on the phenotype of the offspring. Maternal substances are antibodies, hormones, and antioxidants. They shape the immune response and microbiome in young birds. The substances are transferred from mother to chicks by transfer with the yolk sac. This is an important issue in poultry production, where the reproductive cycles are short, and the number of offspring is numerous (Paul et al., 2015).

It has been shown that the quality of nutrition and the breed of chickens affect methylation of the gene encoding the UCP3 (Uncoupling Protein 3) protein in the pectoral muscle (Jin et al., 2018). The UCP3 protein is expressed in skeletal muscle, limiting metabolic regulation at the transcriptional and post-translational levels (Jin et al., 2018). Dietary supplementation with ingredients such as folate, choline, betaine, vitamins B2, B6, B12 increases DNA methylation, because these nutrients donate methyl groups (Jin et al., 2018). The addition of betaine to the feed of broilers increases methylation and thus improves economically important phenotypic features such as meat quality (taste and appearance). Betaine supplementation in newly hatched chickens increases the level of protein in the meat (Murdoch et al., 2016).

The addition of Bacillus subtilis bacteria to the feed and administration in the liquid form of a phytobiotic containing cinnamon oil stimulates the immune system in broiler chickens. It reduces epigenetic changes in the small intestine by inhibiting methyl-glutaryl-coenzyme-A hydroxyreductase, which is involved in the synthesis of cholesterol (Krauze et al., 2020; Ognik et al., 2020). Supplementing the diet with prebiotics increases the amount of beneficial intestinal bacteria Lactobacillus spp. and Bifidobacterium spp. By multiplication, these bacteria increase their colonization area in the caecum, reducing the possibility of colonization by pathogenic microorganisms such as Escherichia coli and Salmonella spp. (Alloui et al., 2013). The dominant bacterial strains in the digestive tract are Firmicutes, Bacteroidetes, and Proteobacteria. These bacterial strains produce bioactive substances, such as biotin, acetate, folate, and butyrate, which mediate epigenetic processes (Zhang, 2015; Morandini et al., 2016). The availability of folic acid directly affects the efficiency of DNA replication and methylation (McKay et al., 2011). Food sources that contain folate are raw leafy vegetables (cabbage, broccoli, spinach). In addition, intestinal bacteria are an additional source of folate. Butyrate is SCFA and a histone deacetylase inhibitor. The production of butyrate depends on the composition of the intestinal microbiota and the presence of Faecalibacterium prausnitzii bacteria, while biotin is responsible for the biotinylation of proteins. This process depends on the constant supply of biotin from the gut microbiota (Verhoog et al., 2019). Biotinylation is an epigenetic mechanism that involves the binding of biotin to histone proteins. Biotinylation of K8 and K12 in histone H4 is dependent on cell cycle. The maximum biotinylation is achieved during mitotic condensation of chromosomes. Histone biotinylation plays a large role in the cellular response to DNA double strand breaks through chromatin structure repair (Shenderov, 2013).

It was reported that the addition of betaine (0.1%) introduced into the broiler (Arbor Acres strain commercial broilers) feed for 44 d changed the CpG methylation pattern in the LPL (lipoprotein lipase) gene by lowering the methylation level in the CpG dinucleotide regions: 1st, 6th, 7th, 8th and 10th to 50th and increasing methylation levels in the 2nd, 5th and 9th dinucleotide regions. As a result of this change, mRNA expression of the analyzed gene was reduced. Phenotypically, birds supplemented with betaine had a better average daily gain and percent abdominal fat than the broilers in the control group (Xing et al., 2011).

Epigenetic changes induced by infection factors

DNA methylation is responsible for silencing transcription, causing changes in the host organism that reduce immunity and increase susceptibility to Marek’s disease (Tian et al., 2013). An experiment carried out on two lines of chickens resistant and susceptible to Marek’s disease (MD) allowed to investigate the epigenetic mechanisms related to the occurrence of the disease (Luo et al., 2012). MD has been shown to alter the expression of methyltransferase (DNMT) genes. Additionally, in chickens susceptible to Marek’s disease, promoters of ALVE and TVB were found to be methylated at the higher level. Hypermethylated genes in MD susceptible chickens are responsible for cell adhesion and the immune response. Marek’s disease virus infection induces the expression level of three methyltransferase genes (DNMT1, DNMT3a and DNMT3b). The level of expression of these genes depends on the life phases of MDV (Luo et al., 2012). There are four phases: an early cytolytic phase 2 to 7 d post-infection, a latency phase of around 7–10 d, a late cytolytic phase that begins on day 18, and a proliferation phase that occurs after day 28. Expression of all three genes is significantly increased in infected chickens during the 5 to 10 d post-infection phase Deregulation of these genes may be crucial in the incidence of Marek’s disease (Luo et al., 2012).

This epigenetic mechanism is involved in immunological processes related to the susceptibility of chickens to Salmonella-induced morbidity The pathogenicity of Salmonella enterica depends largely on its virulence genes, which are called salmonella pathogenicity islands. Salmonella enterica secretes effectors that promote the penetration of bacteria and control host inflammatory responses. For example, the bacterial SipA protein is activated by caspase-3 and plays a key role in bacterial entry into the body. Additionally, the SopE (Salmonella outer protein E) protein promotes the activation of Rho GTPases and initiates the pathogenic bacterial invasion process (Wang et al., 2017). Infection of chickens with the pathogenic strain of Salmonella typhimurium significantly decreased mRNA expression of mucin 2, claudin 1, and occludin in the ileum relative to uninfected chickens (Zhang et al., 2012). It was observed that infection with S. typhimurium resulted in decreased expression of claudin, occludin, and mucin. Reduced expression causes an increase in the permeability of the intestinal barrier of chickens. The intestines damaged in this way have a reduced immune response (Zhang et al., 2012). The studies carried out on two groups of chickens with influenza: the control group and the group infected with Salmonella entericia were aimed at demonstrating epigenetic changes occurring around the start (TSS) and termination (TTA) site of transcription during infection with this bacterium. It was shown that the level of DNA methylation was higher in the genomic regions of TSS and TTA in the S. entericia-infected group (Wang et al., 2017). Genes associated with the immune response of the host organism MHC and MHC class IV show increased methylation following infection with S. entericia. It has been shown that Salmonella infection may result in increased methylation of genes (POLB, PCNA) involved in DNA replication (Wang et al., 2017). The mechanism of disease resistance is determined by DNA methylation, and methylation variability is both a cause and a consequence of an infection in the organism. The results show that DNA methylation can regulate the host immune response by regulating the expression of immune-related cytosine genes in response to infection with S. entericia. The DNA methylation profile of the genome of chickens inoculated with S. entericia was analyzed. It was proved that inoculation of S. entericia was responsible for promoting methylation in the cecum of chickens. The mCHG and mCHH (CHG, CHH regions containing methylcytosine where H corresponding to adenine or tyrosine) methylation decreased in chickens that were inoculated with S. entericia compared to the control group, while the mCpG cytosine methylation increased. In biological processes, methylated cytosine genes are associated with metabolic processes, hormone secretion, and cell aggregation (Wang et al., 2017).

Stress as a trigger of epigenetic modification in chicken

Intensive housekeeping puts chickens under stress. Stressors occurring in the early stage of development of chickens, such as hatching in hatchers, deprivation of maternal contact, transport, and changes in ambient temperature, may influence behavioral and physiological changes, including epigenetic modifications. DNA methylation patterns responsible for silencing gene expression are generally maintained during cell division but can sometimes be modified by external stimuli (Raynal et al., 2012). These modified methylation patterns can be transferred through the germline and remain in somatic cells for future generations (Franklin et al., 2010).

It has been shown that stress induced early in life can affect the nervous system (neural architecture) to make the body resistant to the same stress factor during life (Franklin et al., 2012). One of the biggest challenges in keeping broiler chickens is heat stress. Several experiments have shown the effect of conditioning chicks early by introducing higher temperatures in the henhouse on changes in DNA methylation profile. The research results indicate that thermal conditioning of chickens between 3 and 5 d after hatching showed an improvement in temperature tolerance in 10-d-old chickens due to epigenetic modifications. Yossifoff et al. (2008) conducted a study in which 3-d-old chickens were placed at 37.5 °C for 24 h (conditioning), and then on the 10th day of life, the procedure was repeated (challenge). Tissue from the anterior hypothalamus was collected for research. It was found that the brain-derived neurotrophic factor (BDNF) gene undergoes changes in expression levels during both conditioning and exposure, and these changes coincide with the CpG methylation patterns in the promoter region of the BDNF gene. This suggests that dynamic changes in DNA methylation are involved in the regulation of BDNF gene expression during thermotolerance acquisition. Hatching studies of the level of DNA methylation and histones in chickens subjected to heat stress indicate that epigenetic features differ depending on the environment in which the chickens resided during the hatching period (Kisliouk and Meiri, 2009). It has been proved that the expression of the BDNF gene, which is the key regulator of thermal tolerance in the hippocampus hypothalamus, is different between individuals in the control group and individuals accustomed to higher temperatures in the early stages after hatching. Changes in the methylation level of CpG sites and histone modifications in the BDNF gene promoter were observed during the acquisition of thermal tolerance on the third day after hatching (Kisliouk and Meiri, 2009; Kisliouk et al., 2011). In another experiment, male Cobb broilers were conditioned at mild temperature (36 °C) and high temperature (40 °C) for 24 h. Heat challenge was also used on the 10th day of life in the form of 24-hour exposure to 36°C. Analysis of the genetic material from the hypothalamus, showed that heat conditioning at high temperature, caused increased methylation of CpG accompanied by a decrease in POU Class 2 Homeobox 1 (POU2F1), and binding and an increase in the level of histone H3 acetylation (H3acet) on the heat-shock protein (HSP70) gene promoter. All of these indicates that acute heat stress used for early temperature conditioning may determine the body’s later response to heat stress (Kisliouk et al., 2017).

There was yet another experiment, in which researchers provided first results on how two simultaneous stressors, chronic heat stress and viral infection, affect epigenetic changes. The experimental groups from two different lines of hens (Leghorn and Fayoumi) were subjected to heat stress on the 14th day of life (38 °C for the first 4 h, then 35 °C until the end of the experiment), and additionally, on the 21st day of life, the chickens were inoculated with Newcastle Disease Virus (NDV). In order to compare and characterize the regulatory factors in both chickens’ lines treated with combined stressing factors, bursal tissue was collected in which RNA-seq (gene expression) and ChIP-seq (targeting histone modification) analyses were performed. It was found that stressing factors applied in Leghorn caused downregulations of genes related to cellular processes (cell division and cell cycle). These results might suggest deleterious proliferation of cells in the bursa. Also, the immunity-related gene ontology (GO) terms and pathways associated with regulatory regions with reduced enrichment levels in the histone H3 acetylated at lysine 27 (H3K27ac) and histone H3 monomethylated at lysine (4H3K4me1) modifications explain other potential mechanisms influenced by heat stress and NDV infection. This potential deleterious effect of stress factors on treatment-induced B cells may partly explain why leghorns are more susceptible to both heat stress and NDV (Chanthavixay et al., 2020).

Interesting results regarding the change of methylation patterns in comparison to different conditions of chickens rearing were provided by Pértille et al. (2017). The experiment was carried out on female white Dekalb chickens. At 4 wk of age, the birds were divided and some of them were transferred to aviaries and the other part to cages (stress factor). In this arrangement, the chicks were left until the age of 16 wk, and then, in the 24th week of rearing, blood was collected for laboratory analyzes. DNA methylation analysis was performed using genotyping (GBS) by sequencing and methylated DNA immunoprecipitation (MeDIP). As a result, 115 genomic windows were revealed with significant changes in DNA methylation levels between comparable groups. These changes were located in 53 genes related to immune response, cell signaling related to the MAPK, G protein and opioid pathways, and in 22 intron regions. This potential of using red blood cells to determine epigenetic biomarkers of stress in chickens was also used in other experiments by the same authors. In this case, methylome were compared in red blood cells collected from socially isolated and socially non-isolated animals. The experiment was performed on White Leghorn chickens at two locations (Sweden and Brazil). Between 4 and 26 d of age, randomly selected animals were isolated once a day for 1 h in the first week, 2 h in the second week, and 3 h in the third week. Control animals did not experience isolation. Blood was drawn 2 h after the last stress exposure. The authors reported that after analyzing the differentially methylated regions (DMRs) in the groups of animals exposed to isolation, 7 (Brazil) and 4 (Sweden) significant overlapping DMRs were found (Pértille et al., 2020). These research results could help identify epigenetic biomarkers of stress induced by social isolation in chickens.

Epigenetic Mechanisms in Other Species

Although chicken is very famous among the poultry species, other species such as quails, turkeys, ducks, and geese are also being economically raised all around the world. However, except for quails, these species have been rarely studied with respect to epigenetics. Quails are considered as a very good model to study the epigenetic effects, particularly transgenerational effects in birds because of their relatively short generation interval and easy husbandry (Leroux et al., 2017).

Prehatch environmental factors activating epigenetic mechanisms

The temperature is a major factor of the prehatch environmental and seems to have some epigenetic effects in poultry species—short- and long-term effects. Among those, Wang et al. (2019) showcased the role of epigenetic regulation of genes related to myogenesis expressed in the muscles when the early duck embryos were subjected to a slight increase of the incubation temperature. Here, many genes related to myogenesis were upregulated during the early embryonic phase (Embryonic Day [ED] 1–10) and downregulated during the later embryonic growth (ED 10–20). They studied the methylation status of two genes that were up and downregulated in both cases (ED 1–10 and ED 10–20, respectively). They observed a significant inverse correlation between the methylation status in the promoter region of these genes and their mRNA expression in leg muscles. Therefore, they suggest that these changes of gene expression might be due to epigenetic regulation triggered by the prehatch thermal manipulation. This evidence thereby shows that the thermal regulation during the prehatch period could have an epigenetic effect on muscle growth in ducks in a timely fashion.

In another study of adult Japanese quails, transcriptomic analysis of hypothalamus indicated that the prehatch thermal manipulated quails showed a more prominent response to post hatch heat challenge than control quails via more differentially expressed genes involving mitochondrial and heat-response functions. Therefore, the authors suggest that the prehatch thermal manipulation may have triggered a silent epigenetic gene reprogramming such a way that phenotypic changes will occur only in face of stressful conditions (Vitorino Carvalho et al., 2021). Moreover, using Muscovy ducks and chickens (Tzschentke, 2007), it was further claimed that the warm incubation temperatures induce postnatal heat adaptation while cold incubation temperatures induce postnatal cold adaptation. The author further claims that these adaptations are results of perinatal epigenetic imprinting effects in ducks. Furthermore, in another study of wood ducks (DuRant et al., 2013), it was discovered that the most important factor influencing the ability of ducklings to regulate their body temperature during a post-hatch cold stress was the prehatch temperature as compared to post-hatch feed availability. Nonetheless, Nichelmann (2004) observed no epigenetic imprinting on thermal adaptation in later life in turkeys when the thermal manipulation is performed during the last week of the incubation period, as opposed to positive effects observed in Muscovy ducks. Therefore, it can be suggested that, in poultry, the stage of incubation in which the thermal manipulations will be performed is crucial for triggering the epigenetic imprinting effects on post hatch thermoregulation.

Post-hatch environment factors related to epigenetic regulation

As we mentioned before, the environment where the birds will be grown also greatly affects their phenotype via triggering epigenetic mechanisms (Duncan et al., 2014). In Japanese quails, Wu et al. (2013) observed an increase in egg laying rate by injecting kisspeptin-10 (kp-10) daily for 3 wk. As the mechanisms behind, they discovered that the lipid synthesis in the liver has been greatly altered by kp-10 via up-regulation of many genes, such as SREBP-1, FAS, VLDL-II, CYP7A1, and VTG-II. In another study, it was found that VTG-II gene expression in liver was significantly lower in old female quails that do not lay eggs compared to young female layers (Gupta et al., 2006). They further observed that the -CCGG- region of the promoter region of this gene was hypermethylated and the chromatin was compact in the old female quails in coherent with the expression results. Therefore, it is suggested that epigenetic mechanisms such as methylation play a role in age-dependent reduction of egg laying in poultry species. Similarly, Andraszek et al. (2014) found that the gene encoding the 28S rRNA (RNA28S) in the spermatocytes of male Japanese quails undergo methylation in an age-dependent manner (increased with age). Here, they further suggest that this methylation could be a marker for studying aging processes in animals. Therefore, it can be suggested that the post-hatch environmental stresses causing an aging effect in birds, such as oxidative stress, chronic infection, metallic chemicals, etc. may implicate the epigenetic mechanisms such as methylation in altering the phenotypes of poultry species.

Some other studies observed epigenetic effects through artificial treatments such as injections of chemicals, infection with pathogens, and manipulating the dietary supplements. Fehrer et al. (1988) claimed that circulating prolactin level in turkeys may be implicated by DNA methylation where they observed increased prolactin level due to injecting a cytosine methylation inhibitor, 5-Azacytidine to sexually immature ducks. In another study, CD8A gene, which is crucial in cell mediated immunity, was highly downregulated in the ducklings infected with duck hepatitis virus type 1 (DHV-I) (Xu et al., 2014). They claimed that the mechanism behind this modification is the methylation in CpG sites of the promoter region of this gene. Although the exact mechanism between CD8A gene expression and DHV-I is not well elucidated, the authors suggest that CD8A gene could be used as an epigenetic biomarker for selection against DHV-I infection. Dietary supplementation of genistein, a methylation modifier, caused positive effects such as higher feed intake, efficiency, egg production, egg weight, Haugh unit, and shell thickness in Japanese quails (Akdemir and Sahin, 2009). This indicates that these traits are largely affected by the epigenetic effects in birds. Dietary supplementation of betaine and methionine in geese also affected the transcription regulation of hepatic genes via reduction of the methylation level in LOC106032502 in liver (Yang et al., 2018).

There is an evidence for aging-related histone modifications also among the poultry species. Mishra and Kanungo (1994) and Mahendra and Kanungo (2000) described the tissue specific histone modifications in Japanese quails which are influenced by the aging process. They observed these modifications in liver and oviduct, to be differential when different steroid hormonal induction treatment was given to the young, adult, and old quails.

RNA-based environmental epigenetic effects in poultry species were mainly observed with the infection of various pathogens. Liu and co-workers have identified a differential expression pattern of miRNA expression during the infection of highly pathogenic avian influenza virus (HPAV) H5N1 in ducks who are immune to the infection (Li et al., 2015). This finding provided confirmation for a possible role of miRNA in the immunity against HPAV infection in birds. Moreover, Samir et al. (2020) found organ-specific miRNA expression in lungs and brain of HPAV-H5N1–infected ducks. The variations in miRNA expression were attributed to differences in enriched pathways providing insights into differential replication of HPAV and organ damage during the infection. Furthermore, miRNA expressions in duck liver during duck hepatitis A virus type 3 (DHAV-3) have been studied by Wu et al. (2020) and identified miRNA-based regulation of important genes of immune-related pathways in liver. Noncoding RNAs (miRNA, cirRNA, lncRNA)–based differential gene regulation was also observed in the Salmonella enteritidis infection in duck granulosa cells, indicating candidate regulators of the infection in ducks (Zhang et al., 2021).

Apart from the roles in immunity, miRNA seems to regulate the lipid metabolism in the liver of ducks when fed with varying proportion of fat in diet (He et al., 2016). Additionally, green eggshell production in ducks, which may occur in response to environmental changes, seems to be epigenetically regulated via miRNA-based mechanisms within the duck shell glad tissues (Xu et al., 2018).

Transgenerational epigenetic effects

Leroux et al. (2017) showed the transgenerational epigenetic effects in Japanese quails injected with the methylation modifier genistein into eggs. Effects were observed in the 3rd generation where the Genistein treated birds showed lower body weight, lower egg production, and interestingly lower distress to isolation, compared to control group. These results suggest that epigenetic effects on both production and behavioral traits could be trangenerationally transmitted in Japanese quails. Nevertheless, these results are contradictory to the positive effects observed in (Akdemir and Sahin, 2009) upon dietary supplementation of genistein to Japanese quails. However, they observed the effects on the same generation while Leroux et al. (2017) observed transgenerational effects. Therefore, these studies indicate that the same environmental effect may involve different mechanisms depending on the generation and the mode of exposure in the birds.

Summary and future perspectives

A great advantage in the field of poultry epigenetics is the fact that there is possibility of manipulation during embryonic development, early programming, and stimulation. The current epigenetic challenges are in the area of supplementation of the organism and the stimulation of the intestinal microbiota, as well as epigenetic inheritance. Understanding the common interaction between the host and its microbiota will provide a better understanding of the epigenetic modifications taking place, which can be used to determine their role in shaping the health status of poultry. An important direction of the analysis is also the possibility of targeted and stable silencing of the expression of genes important for the physiology of the host.

Glossary

Abbreviations

miRNA

microRNA

PGCs

primordial germ cells

ECS

chicken embryonic stem cells

RRBS

reduced representation bisulfate sequencing

IOF

in ovo feeding

mtDNA

mitochondrial DNA

MD

Marek’s disease

DNMT

methyltransferase

MeDIP

methylated DNA immunoprecipitation

ChIP-Seq

, chromatin immunoprecipitation assay

DMRs

differentially methylated regions

ED

embryonic day

DHV-I

duck hepatitis virus type 1

HPAV

avian influenza virus

cirRNA

circular RNA

lncRNA

long non-coding RNA

CHG

CHH, regions containing methylcytosine where H corresponding to adenine or tyrosine

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.