Studying the role of chicken gut derived Lactic Acid Bacteria (LAB) Supplementation on

Chicken Growth, Gut Health, Epigenetics (Histone lactylation), and Molecular Responses

Research Objectives:

Growth Performance and Meat Quality:

 Assess the influence of LAB supplementation on chicken growth performance.

 Investigate the effects of LAB on muscle development and meat quality in broiler

chickens.

Gut Health and Microbiota:

 Examine the impact of LAB on gut health, including intestinal morphology, enzyme

activity, and barrier function.

 Analyze changes in the chicken gut microbiota composition due to LAB

supplementation.

Epigenetics and Molecular Mechanisms:

 Explore the effects of LAB supplementation on histone lactylation patterns in chicken

tissues.

 Understand the interplay between LAB supplementation, gut microbiota, and epigenetic

regulation in chickens.
Introduction:

Lactic Acid Bacteria (LAB) are a group of Gram-positive bacteria that produce lactic acid as

a major end product of carbohydrate fermentation. LAB are widely used as probiotics, which

are live microorganisms that confer health benefits to the host when administered in adequate

amounts. LAB have been shown to modulate the intestinal microbiota of poultry, which is a

complex and dynamic community of microorganisms that plays a vital role in digestion,

immunity, and metabolism. LAB can improve poultry gut health by enhancing intestinal

barrier function, reducing inflammation, inhibiting pathogens, and modulating immune

responses. LAB can also improve poultry growth performance and meat quality by

increasing feed efficiency (FE), average daily gain (ADG), average daily feed intake (ADFI),

and feed conversion ratio (FCR), as well as improving muscle development, lipid profiles,

and meat texture. LAB can affect the epigenetic regulation of gene expression (Chen et

al., 2022; Zhang et al., 2019)in poultry tissues, such as muscle and liver, by altering histone

lactylation patterns. Histone lactylation is a novel type of histone modification that involves

the addition of lactate to lysine residues on histone proteins. Histone lactylation can

influence gene expression by changing the chromatin structure and accessibility, as well as

recruiting or repelling transcription factors and chromatin modifiers. Histone lactylation can

be affected by environmental factors, such as diet, stress, and microbiota. Therefore, LAB

supplementation may have an impact on histone lactylation levels in poultry tissues,

which may in turn affect gene expression patterns related to growth, development,

metabolism, and immunity.

Histone Lactylation:
Addition of a lactyl group to lysine residues in the histone tails is a unique histone

modification known as histone lactylation. It is created during the process of glycolysis from

the molecule lactate. A lactyl group is added to lysine residues in the histone tails during the

unique histone post-translational modification known as histone lactylation. It derives from

lactate, a molecule produced during glycolysis under either aerobic or anaerobic

circumstances (Liu et al., 2022). Numerous biological processes, including metabolic

balance, immunological responses, inflammation, and carcinogenesis, have been linked to

histone lactylation. Histone lactylation is regulated by the balance between lactate production

and consumption, as well as by the activity of lactate dehydrogenase and sirtuins (Chen et al.,

2022; Liu et al., 2022; Zhang et al., 2019). Histone lactylation affects gene expression by

altering the chromatin structure and recruiting or repelling transcription factors and

chromatin modifiers. Histone lactylation is a dynamic and reversible modification that

reflects the metabolic state of cells and tissues, and plays a key role in epigenetic regulation

of health and disease (Dai & Lv, 2023; Zhang et al., 2019).

Histone lactylation has been connected to numerous biological functions in multiple

organisms, including metabolic equilibrium, immunological responses, and inflammation.

However, a lot is to be understood about how it specifically responds to lactic acid

metabolism (Sgarra et al., 2023)or gut bacteria, particularly in avian species like chickens.

Understanding how histone lactylation works as an epigenetic regulator of chicken

adaptation may help us better understand the molecular processes driving avian muscle

growth and meat quality. This study proposal's primary goal is to ascertain the impact of

LAB supplementation on chicken histone lactylation and gene expression patterns. We'll also

investigate any functional effects that histone lactylation may have on the physiology and
behavior of chickens. By doing so, we aim to elucidate the role of histone lactylation in

epigenetic regulation of chicken.

How lactic acid bacteria (LAB) in gut might change Histone lactylation patterns?

By changing the metabolic balance of lactate and pyruvate in the host cells, lactic bacteria

can modify histone lactylation. The primary mechanism for the breakdown of glucose is

called glycolysis, and it involves the interconversion of two metabolites called lactate and

pyruvate. The fermentation of glucose by lactic bacteria can result in the production of lactic

acid, which can reduce pH and raise the level of lactate in the host cells (Xin et al., 2022).

Lactate dehydrogenase (LDH), which breaks down NADH and generates NAD+, can turn

lactate into pyruvate. Sirtuin deacylases (SIRTs), which may remove lactyl groups from

histones and other proteins, require NAD+ as a cofactor. Therefore, lactic bacteria can

modify the availability of NAD+ and SIRTs as well as the ratio of lactate to pyruvate in

the host cells to impact histone lactylation (Izzo & Wellen, 2019; Xie et al., 2022). On the

one hand, lactate regulates intracellular and extracellular metabolic processes across the

entire body. On the other hand, it also has a variety of biological effects, including anti-

inflammation, immunological regulation, and gene expression, through receptors expressed

in different cells and tissues (Brooks, 2020). Recent findings by Zhang et al. state that lactate

contributed to epigenetic regulation of genes by lactylating histone lysine residues and that

lactate was found to be a precursor to histone lysine lactylation (Kla), which stimulated gene

transcription from chromatin (Zhang et al., 2019).

Research Question raised from previous studies on Histone lactylation?

Previous studies have reported that addition of lactyl group to histone is mainly triggered by

lactate accumulation. But it has largely remained unclear that histone lactylation is only

consequence of lactate accumulation. We are not sure whether lactate concentration in the

nucleus causes histone lactylation. In this study, we can address this question that whether

availability of dietary lactate from lactic acid bacteria can induce histone lactylation or

trigger other metabolic changes in chicken.

References:

Chen, L., Huang, L., Gu, Y., Cang, W., Sun, P., & Xiang, Y. (2022). Lactate-Lactylation

Hands between Metabolic Reprogramming and Immunosuppression. In International

Journal of Molecular Sciences (Vol. 23, Issue 19). MDPI.

https://doi.org/10.3390/ijms231911943

Dai, X., & Lv, X. (2023). Lactate, histone lactylation and cancer hallmarks. In Expert

Reviews in Molecular Medicine. https://doi.org/10.1017/erm.2022.42

Izzo, L. T., & Wellen, K. E. (2019). Histone lactylation links metabolism and gene

regulation. In Nature (Vol. 574, Issue 7779, pp. 492–493). NLM (Medline).

https://doi.org/10.1038/d41586-019-03122-1

Liu, X., Zhang, Y., Li, W., & Zhou, X. (2022). Lactylation, an emerging hallmark of

metabolic reprogramming: Current progress and open challenges. In Frontiers in Cell and

Developmental Biology (Vol. 10). Frontiers Media S.A.

https://doi.org/10.3389/fcell.2022.972020
Sgarra, R., Battista, S., Cerchia, L., Manfioletti, G., & Fedele, M. (2023). Mechanism of

Action of Lactic Acid on Histones in Cancer. Antioxidants & Redox Signaling.

https://doi.org/10.1089/ars.2022.0190

Wang, L., Lin, Z., Ali, M., Zhu, X., Zhang, Y., Li, S., Li, K., Kebzhai, F., & Li, J. (2023).

Effects of lactic acid bacteria isolated from Tibetan chickens on the growth performance and

gut microbiota of broiler. Frontiers in Microbiology, 14.

https://doi.org/10.3389/fmicb.2023.1171074

Xie, Y., Hu, H., Liu, M., Zhou, T., Cheng, X., Huang, W., & Cao, L. (2022). The role and

mechanism of histone lactylation in health and diseases. In Frontiers in Genetics (Vol. 13).

https://doi.org/10.3389/fgene.2022.949252

Xin, Q., Wang, H., Li, Q., Liu, S., Qu, K., Liu, C., & Zhang, J. (2022). Lactylation: a Passing

Fad or the Future of Posttranslational Modification. In Inflammation (Vol. 45, Issue 4).

https://doi.org/10.1007/s10753-022-01637-w

Zhang, D., Tang, Z., Huang, H., Zhou, G., Cui, C., Weng, Y., Liu, W., Kim, S., Lee, S.,

Perez-Neut, M., Ding, J., Czyz, D., Hu, R., Ye, Z., He, M., Zheng, Y. G., Shuman, H. A.,

Dai, L., Ren, B., … Zhao, Y. (2019). Metabolic regulation of gene expression by histone

lactylation. Nature, 574(7779). https://doi.org/10.1038/s41586-019-1678-1

Brooks, G.A. 2020. Lactate as a fulcrum of metabolism. Redox Biology 35:101454.

 Experimental Design

1. Sample Collection and (Lactic acid bacteria) LAB Isolation:

Cecum content from 40-50 yellow feathered chickens will be collected immediately, frozen

in liquid nitrogen, and stored at −80°C until further analysis.

Bacterial culture and purification will be carried out.

The isolated strains will be identified by gram staining, biochemical tests, and 16S rRNA

sequencing.

Microbial genomic DNA will be extracted using established methods, and the 16S rRNA

gene will be amplified for identification. (Wang et al., 2023)

2. In vitro Experiments:

Conduct in vitro antibacterial and acid tolerance experiments using isolated LAB strains to

assess their potential beneficial properties.

3. Animal Trials:

Total 240 chickens will be reared for the experiment with each group having 6 replicates, and

each replicate will have 10 chickens.

Randomly divide the chickens into experimental groups.

4. Experimental Groups:

Commercial chicken will be purchased with similar initial weight and will be given

acclimation period of three days. Experiment will start after 3 days and will end at 28 days.

A. Control Group (CON): Raised at basal diet without LAB supplementation.

B. Treatment Groups (T1, T2, and T3): Supplemented with different concentrations of

Lactic Acid Bacteria (LAB) isolated from chicken gut.

Administer a basal diet to one group and the same diet supplemented with different

concentrations of LAB (e.g., 100 mg/kg, 200 mg/kg, and 400 mg/kg) to the other groups for

a specified period (e.g., 42 days).

Sample Collection and procedures to be performed:

Performance and Growth:

 Assess parameters such as Average Daily Gain (ADG) and Average Daily Feed Intake

(ADFI).

 Record daily feed intake and body weight.

 Calculate feed conversion ratios (FCR) and average daily gain (ADG).

Physiological Parameters:
 Assess physiochemical parameters during the experiment to monitor chicken health.

Serum Parameters:

 Measure serum levels of ghrelin, insulin-like growth factor-1 (IGF-1), and growth

hormone (GH).

Histone Lactylation:

 Isolate histones from collected tissues (e.g., muscle, liver).

 Perform histone lactylation profiling using mass spectrometry.

Muscle Growth:

 Conduct muscle biopsies to assess muscle fiber size, composition, and connective tissue

deposition.

Meat Quality Assessment:

 Evaluate meat attributes, including color, texture, moisture content, and lipid profiles.

Gut Health:

 Collect fecal samples to assess gut microbiota composition using 16S rRNA gene

sequencing.

 Analyze gut health markers (e.g., gut barrier integrity, inflammation).

 Extract metagenomics DNA from cecal digesta.

 Perform metagenome assembly, binning, and taxonomic assignment.

 Analyze microbial co-occurrence networks to understand community interactions.

 Quantify short-chain fatty acids (SCFAs) in cecal digesta using gas chromatography.

Transcriptomic and Gene Expression:

 Extract RNA from tissue samples (e.g., muscle, liver) collected at end of experiment.

 Perform transcriptomics analysis using next-generation sequencing techniques (RNA-

Seq) to assess gene expression patterns in different metabolic, glycolytic and growth

related pathways.

 Analyze differentially expressed genes (DEGs) to identify genes affected by LAB

supplementation.