Gut health and gene expression in poultry

Microarray technologies have spurred the development of the field of nutrigenomics. Using microarrays to evaluate the activity of thousands of genes simultaneously, offering a rapid measure of nutrient-induced, physiologic changes. Traditional animal trials involve feeding a specific diet and measuring whole-animal changes such as growth rate, production or performance. These traditional approaches are labor-intensive, time-consuming and require large numbers of animals to achieve statistical differences. A nutrigenomics approach allows us to use smaller numbers of animals and generate a multitude of data in a short time. Additionally, nutrigenomics now provides us with tools to understand the molecular and cellular changes that cause changes to the whole animal.

Genes encode the proteins that are the building blocks for all cellular structures. Because cells are part of a dynamic life cycle (i.e., birth, growth, death), proteins are constantly being manufactured (synthesized) and then broken down. Cells regulate which proteins will be produced by starting or stopping transcription. Transcription is the first step in the gene expression process, a process that takes information from the gene to make a protein or other gene product for cell use. Gene transcription is highly regulated; not only can it be turned on or off, but also the amount of transcription can be increased or decreased in response to a stimulus. When we look at data from a microarray ("gene chip") we can see the downstream effect of particular nutrients on the subcellular level. When a nutrient is absorbed and metabolized, it affects different components of the cell. For example, a nutrient may result in the production of an altered protein, which in turn results in a "cascade." A cascade is a series of proteins or other molecules that interact and change the structure of a protein. Such changes can activate or inhibit gene transcription by altering a protein's ability to interact with RNA polymerases, the enzymes that produce RNA.

The gene chip is the tool that allows us to identify which genes respond when our product is fed to a type of animal. We set up a typical experiment by comparing a control group of animals to a treatment group of animals. Animals in both groups are of the same breed, size, age and sex to ensure they are as genetically alike as possible. Next we make sure that the animals are consuming the same diet with the exception of the nutrient of interest. For instance if we were interested in looking at selenium, we would feed the control group a diet with no added selenium; the treatment group would be fed that same diet but with selenium added. Now we can attribute any differences we see at the gene level between the two groups to the presence or absence of dietary selenium. The gene chip allows us to measure the nature and amount of mRNA genes produce. Using gene chips, we can look at specific genes of interest or at multiple genes that define whole cascades or pathways that are associated with physiologic changes such as inflammation or oxidative damage.

The following studies were the first to examine the effects of Se source on gene expression profiles in avian reproductive tissues. The key findings from these studies suggest that specific transcriptional profiles in avian reproductive tissues differ depending on the form of Se fed, which may explain the parallel reproductive differences we see in whole animals.

Selenium (Se) has long been known to play a key role in both male and female reproduction. Selenium deficiency leads to reproductive disorders causing reduced fertility in females. In hens, Se deficiency leads to reduced egg production in both broilers and layers. In males, even moderate Se deficiency results in abnormal sperm and decreased sperm motility. Long-term Se deficiency leads to changes in sperm numbers and decreased fertilizing capacity. Reversal of these reproductive abnormalities in both males and females with Se supplementation confirms a key role for Se in reproduction; however, relatively little is known about the mechanisms by which Se exerts its physiologic effects in reproductive tissue.

To elucidate the molecular mechanisms behind the Sel-Plex® effects on reproductive function, two studies were conducted using flocks maintained on long-term diets with or without supplemental Se (Sel-Plex or sodium selenite).

In the female reproductive study, broiler breeder hens were fed semi-purified diets with or without supplemental Se (Brennan et al., 2011a). At 49 weeks of age, seven hens from each treatment (control, Sel-Plex, or sodium selenite) were euthanized and oviduct tissue was sampled. Tissue Se content and gene expression patterns were analyzed to see whether molecular changes could shed light on changes seen in a whole-bird egg production. Tissue levels from the two Se treatment groups did not differ and supplementation with either form of Se resulted in greater oviduct Se accumulation compared with levels from the control birds. However, using commercially available chicken genome microarrays, analysis of transcriptional profiles revealed vast differences between the two Se treatment groups. Overall, 2106 (1961 down-regulated, 145 up-regulated) genes were differentially expressed in sodium selenite supplemented hens and 947 (639 down-regulated, 308 up-regulated) in Sel-Plex-supplemented hens. Critical genes involved in oxidative phosphorylation, ATP production and protein translation were up-regulated by Sel-Plex but down-regulated by sodium selenite compared with control hens. In addition, two selenoproteins that are thought to play key roles in male reproduction, GPX4 and Sepp1, were up-regulated by Sel-Plex® but not sodium selenite.

In the male reproductive study, layer roosters were fed a corn-soy diet with or without supplemental diets (Brennan et al., 2011b). At 40 weeks of age, seven roosters from each treatment (control, Sel-Plex® or sodium selenite) were euthanized and testis tissue was sampled, weighed and frozen. Although testes weight did not differ between treatments, roosters fed Sel-Plex had a great testicular Se content than control or sodium selenite birds. Analysis of the gene expression profiles from control, sodium selenite and Sel-Plex roosters revealed that both Se supplementation and Se source were associated with changes in mRNA transcripts in testes compared with control roosters. Similar to responses seen in the oviduct, we found that testis tissue from birds supplemented with Sel-Plex had a greater number of differentially expressed genes than birds supplemented with sodium selenite. Overall, Sel-Plex supplementation altered expression of 442 genes (318 up-regulated, 124 down-regulated) compared with the control. In sodium selenite supplemented birds, 155 genes were differentially expressed in the testes (93 up-regulated, 62 down-regulated) compared with the control.

Using pathway analysis software, we found that Se supplementation, regardless of source, affects the expression patterns of genes involved in testis cell structure and morphology. Although both sources of Se affected genes that had similar biofunctions such as cellular development, Sel-Plex supplementation led to a greater enrichment than sodium selenite (103 affected genes versus 11 affected genes, respectively). In addition, Sel-Plex supplementation appeared to affect canonical pathways that play important roles in proper male reproductive function. These pathways included integrin-linked kinase (ILK) and actin cytoskeleton signaling pathways. The proper function and turnover of specialized actin-related cell-cell junctions in the seminiferous epithelium is fundamental to normal spermatogenesis

A healthy gut is a key factor in the efficient utilization of feed and therefore in overall animal performance. Actigen is a yeast cell wall derivative used in broiler diets to promote growth through immune modulation and improved intestinal health. In this study, we reported differences resulting from supplementation with Actigen™ or bacitracin methylene disalicylate (BMD), an antibiotic growth promoter, on animal performance and corresponding changes in gut cell morphology. When gene expression profiles were studied, we found that genes involved in protective functions and pathways were influenced by Actigen and/or BMD. Finally, we saw a corresponding change in mucin 2 mRNA levels that corresponds to changes in goblet cells and reflects a change in gut health.

Previous research has found that supplementing broilers with Actigen or BMD results in improved weight gain and feed conversion ratios (Collett et al., 2011). To clarify the molecular mechanisms behind Actigen's effects on animal growth and performance, broilers were fed a standard commercial diet supplemented with Actigen or BMD for 6 weeks. At the end of the 6-week period, 7 birds from each treatment were euthanized and the jejunum was collected for histologic and gene expression analysis. Analysis of tissue morphology indicated that supplementation with either Actigen or BMD resulted in an increased goblet cell villus height and an increased villus height to crypt depth ratio in the jejunum. We also found that overall goblet cell number increased with either Actigen or BMD. To understand how these products commonly, or differentially, affect gut health and improve animal performance, we used gene expression analysis to help understand their cellular effects.

Microarray analysis revealed differences in gene expression patterns in the jejunum from birds supplemented with Actigen or BMD (Brennan et al., 2011c; Xiao et al., 2011). When birds were supplemented with Actigen, 928 genes were significantly changed (P ≤ 0.05, FC ≥ 1.2; 456 down-regulated, 472 up-regulated) while BMD supplementation resulted in 857 genes that significantly changed (408 down-regulated, 449 up-regulated). Surprisingly, we found that 316 genes were commonly significantly changed by Actigen and BMD (146 down-regulated, 170 up-regulated).

When differentially affected genes were grouped by biologic function, we found that Actigen and BMD commonly altered genes involved in antimicrobial response, inflammatory response, and infection mechanism, cell-to-cell signaling and interaction, and cellular development. Pathway analysis suggests that Actigen supplementation resulted in the up-regulation of signaling pathways involved in cellular immune response, inflammatory response and antimicrobial protection. In addition, we confirmed with real-time PCR that mucin 2 mRNA levels tend to be greater in Actigen- and BMD-supplemented birds compared with the control (Brennan et al., 2010). The mucin 2 gene encodes a protein component of the mucosal layer that protects the intestinal cell wall from bacterial translocation.

Results from these two sets of studies demonstrate that nutrigenomics provides potent tools to analyze the biologic function and molecular mode of action of specific nutrients. The information we obtain from nutrigenomics studies provides a wealth of knowledge that can help us understand the role of proper nutrition on performance, health and disease in agricultural animals. When we incorporate our molecular findings with information from whole-animal studies, such as weight gain or egg production, we can better understand how nutrients specifically elicit their effects on animal health and production.

Brennan, K.M., C.A. Crowdus, A.H. Cantor, A.J. Pescatore, J.L. Barger, K. Horgan, R. Xiao. R.F. Power and K.A. Dawson. 2011a. Effects of organic and inorganic dietary selenium supplementation on gene expression profiles in oviduct tissue from broiler-breeder hens. Anim Reprod Sci. 125:180-188.

Brennan, K.M., J.L. Pierce, A.H. Cantor, A.J Pescatore and R.F. Power. 2011b. Source of selenium supplementation influences testes selenium content and gene expression profiles in the testes of Single Comb White Leghorn roosters. Bio Trace Elem Res (in print).

Brennan, K.M., G.F. Mathis, R. Xiao, B.S. Lumpkins, and J.L. Pierce. 2011c. Comparison of Actigen™ and bacitracin methylene disalicylate (BMD) supplementation gene expression profiles in the jejunum of 6-week old broilers. 2011 PSA national meeting.

Brennan, K.M., T. Ao, and J.L. Pierce. 2010. Effects of Actigen™ supplementation on mRNA levels of mucin and markers of gut health in the jejunum of broiler chicks. J Anim Sci. 88 (E-Suppl 2): 650.

Collett, S.R., G.F. Mathis, B. Lumpkins, D.M. Hooge, K.M. Brennan and J.L. Pierce. 2011.Live performance and intestinal morphology of broiler chickens fed diets supplemented with BMD®, Actigen® or neither product in two pen trials on built-up litter. 2011 PSA national meeting.

Xiao, R., R.F. Power, D. Mallonee, L. Spangler, K.M. Brennan, J. Pierce and K.A. Dawson. 2011. Gene expression study reveals the association of dietary supplementation of ActigenTM and the regulation of pathogen-influenced signaling pathways in broiler chickens. 2011 PSA national meeting