Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Poultry Science
Non Technical Summary
It has been well documented that microbiota acquired in early life plays an outsized role in establishing the gut health trajectory in later life. Therefore, approaches to manipulate and "seed" the early gut microbiota, including probiotic usage, have been of great interest. To date, no cost-effective microbiota-modulators have been discovered that can replace antibiotic growth promoters (AGPs). Furthermore, future solutions need to consider both human health concerns (antibiotic-resistant strains), and social considerations (ethical treatment of animals, welfare) even as we strive towards sustainable animal production.Therefore, there is a desperate need for solutions that offer scientifically-backed solutions that are economical and scalable. Modulation of the gut microbiota colonization by photoperiods, or using monochromatic light is, therefore, potentially important and crucial avenues. And especially if gut microbiota profiles modulated this way can be used as a means to control or influence the outcomes of intestinal infections, such as necrotic enteritis.In our previous work, we showed that chicken raised under different photoperiods acquire significantly different microbiota. However, that study only examined the first three weeks of life - a period where colonization of the gut is ongoing. However, our previous work did not answer whether such differences in microbiota are sustained beyond the early life, colonization period and if so whether they translate into benefits for fighting against intestinal pathogens. In this current study, we will compare the trajectories of broiler gut microbiota beyond the first three weeks and will span almost the entire life of modern broilers leading up to slaughter (7-8 weeks).
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
A healthy gut microflora is recognized for its crucial role in maintenance of gut health, metabolic, and immune homeostasis. As poultry production has moved to antibiotic-free models, approaches to sustain gut health are desperately needed. One of the long term goals of our research program is to enable innovative solutions for improved poultry health through modulation of the gut microbiota. Towards this end, we have conducted studies to evaluate the role of photoperiods in establishing and maintaining beneficial gut microflora. Our short term goals are to identify photoperiod regimens and monochromatic light as a means to promote the colonization of beneficial gut microbiota. This is based on emerging studies, and also our preliminary data, which show that normal photoperiods promote strong circadian rhythms, and enable colonization by beneficial bacteria, compared extended photoperiods. Our central hypothesis is that photoperiods and light frequencies can be used to modulate the membership of the microflora early in life, which sets the tone for improved gut health in later life. To address this hypothesis, we will test two specific objectives.Objective 1: To characterize the gut microbiota under normal versus extended photoperiods, in association with necrotic enteritis occurrence in broilers. We will raise three replicate broiler flocks under two photoperiod regimens and characterize cecal microbiota using 16s rRNA amplicon sequencing.Objective 2: To characterize gut microbiota colonization in broilers exposed to monochromatic light. Hatch-day broilers will be raised under white versus blue monochromatic light in 12-hour light-dark photoperiods to determine their influence on gut microbiota colonization and diversity. We will also evaluate these treatments and their relationship to occurrence of necrotic enteritis.
Project Methods
We will achieve our objectives through a combination of live-animal trials under different photoperiods, in combination with molecular approaches to characterize gut microbiota membership, as well as occurrence of necrotic enteritis. The live animal studies, sampling of birds for microbiota analysis will be designed, coordinated and carried out by the Athrey lab (PI). Tissues collected for analysis of necrotic enteritis diagnosis will be carried out by the lab of Dr. Audrey McElroy (co-I). All live-animal work will be carried out at the Texas A&M Poultry Research and Education center, whereas molecular and computational work will be carried out in the department of poultry science labs at the Kleberg building.If as we hypothesize, the colonization of beneficial microbiota can be modulated by photoperiods, we would expect to see a) significantly different microbiota communities between normal (beneficial) and extended photoperiods (not-beneficial), and b) that beneficial gut microbiota composition is associated with lower incidence of necrotic enteritis.Objective 1: To characterize the gut microbiota under normal versus extended photoperiods, in association with necrotic enteritis occurrence in broilers. Study design: We will raise two replicate flocks of 36 commercial broilers for each of three photoperiod treatments, namely - 12L/12D, 18L/6D, and 23L/1D, starting with hatch-day old chicks. The birds will be placed on identical ad libitum feed (commercial broiler feed), on a three phase diet (starter, grower finisher). Daylight conditions will be replicated by using dimmable LED lamps. The beginning and end of a light period, the lights will be dimmed to 500 lux for 10 minutes prior to complete darkness, while daylight intensity at 1300 lux. We will sample birds from three time points (3 week, 5 week and 7 week), to assay the gut microbiota and to assess occurrence of necrotic enteritis. Ten birds from each replicate pen will be euthanized at every sampling time points (10 birds x 2 replicates x 3 treatments x 3 time points = 180 birds) and included in analyses. With this approach we will have 20 individual microbiota profiles per treatment and time point, which will provide us with sufficient power to distinguish community differences (40).Microbiota analyses: We will isolate DNA from cecal content using the MoBio PowerFecal kit according to the manufacturer's instructions. 20 ng of purified DNA will be used for PCR amplification of bacterial 16S rRNA gene sequences, using Q5â High-Fidelity DNA polymerase (NEBNextâ High-Fidelity 2X PCR Master Mix, New England BioLabs, Ipswich, MA). A nested PCR design will be used to amplify the V4 variable region of the 16s rRNA gene, followed by addition of Illumina sequencing primersand Illumina indexes. The amplicons will be sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA) using the paired-end mode. Raw data will be processed and analyzed using the Mothur tool (41) for microbiota analysis.Necrotic Enteritis diagnosis: Diagnosis of necrotic enteritis will be performed on tissue from the small intestine (ileum/jejunum). Diagnosis will be based on presence of gross lesions, and gram stained smear of the mucosal lining. As these tissues will be collected upon planned euthanasia, we can assure that the tissue and lesions are not artefacts of degradation (42). These diagnoses will be performed by the lab of Dr. McElroy.Objective 2: To characterize gut microbiota colonization in broilers exposed to monochromatic light. Study design: We will raise two replicate flocks of 36 commercial broilers for each of three light treatments, namely - standard white lighting at 1300 lux, and blue light (450-480nm), both on a 12/12 LD rotation, and a third blue light treatment with a photoperiod of 18/6 LD. It has been shown that blue light helps chicken stay calm, and may provide welfare benefits (34). The birds will be placed on identical ad libitum feed (commercial broiler feed), on a three phase diet (starter, grower finisher). We will sample birds from three time points (3 week, 5 week and 7 week), to assay the gut microbiota and to assess occurrence of necrotic enteritis. Ten birds from each replicate pen will be euthanized at every sampling time points (10 birds x 2 replicates x 3 treatments x 3 time points = 180 birds) and included in analyses. With this approach we will have 20 individual microbiota profiles per treatment and time point, which will provide us with sufficient power to distinguish community differences (40).Analysis of circadian rhythms: From the euthanized birds, we will isolate peripheral brain tissue to assay the expression of circadian genes (CLOCK, BMAL1, and PER3) to determine if white and blue light are equivalent in their ability to reinforce circadian rhythms, and hence have a similar basis for regulating host homeostasis. Microbiota analyses: We will isolate DNA from cecal content using the MoBio PowerFecal kit according to the manufacturer's instructions. 20 ng of purified DNA will be used for PCR amplification of bacterial 16S rRNA gene sequences, using Q5â High-Fidelity DNA polymerase (NEBNextâ High-Fidelity 2X PCR Master Mix, New England BioLabs, Ipswich, MA). A nested PCR design will be used to amplify the V4 variable region of the 16s rRNA gene, followed by addition of Illumina sequencing primersand Illumina indexes. The amplicons will be sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA) using the paired-end mode. Raw data will be processed and analyzed using the Mothur tool (41) for microbiota analysis.Necrotic Enteritis diagnosis: Diagnosis of necrotic enteritis will be performed on tissue from the small intestine (ileum/jejunum). Diagnosis will be based on presence of gross lesions, and gram stained smear of the mucosal lining. As these tissues will be collected upon planned euthanasia, we can assure that the tissue and lesions are not artefacts of degradation (42). These diagnoses will be performed by the lab of Dr. McElroy.Analysis of data:Following generation of data on Necrotic Enteritis occurrence, and microbiota profiles, we will carry out multivariate analyses to determine if and how microbiota membership are associated with enteritis outcomes. Example analyses will include the estimating the odds that the presence or absence of a certain taxon (or Operational Taxonomic Unit) is associated with risk of necrotic enteritis. We will use a model selection approach to determine whether the presence/absence, or the abundance of specific taxa are influential on NE outcomes. Finally, we will also test the hypothesis of whether necrotic enteritis selects for a certain microbiota community, which would indicate that occurrence of NE influences microbiota structure instead of vice versa. Our temporal data will help resolve if and when such changes are occurring.