Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
Animal Science
Non Technical Summary
Over the last few decades, there has been an increasing demand for poultry meat as a good protein source in the diet. To meet this demand, the poultry industry initiated several strategies to increase broiler meat production. One among them was the inclusion of antibiotics in the broiler diet. In effect, use of antibiotics as growth promoters (AGPs) helped improve bird health, growth and performance. However, use of antibiotics as feed additives led to concerns of antibiotic resistant pathogens and their implications to human health. Consequently, the FDA has curbed the use of AGPs in food animals including poultry. This restriction led to problems with performance, increased production costs and rise in diseases in poultry. Therefore, there is a need to develop effective alternatives to help promote health and performance in broilers. Among the different alternatives tested, feeding probiotics to chickens was shown to improve body weight gain and feed conversion ratio in chickens. In modern-day broilers, the period of embryonic growth accounts for almost half of the lifespan. Also, this period of development is critical to chick performance following hatch. Hence, any approach that can support and promote embryo growth is expected to have a positive impact on performance in broilers. Specifically, this study will apply probiotics on eggs and study their effect on embryonic growth and performance in broilers. Additionally, this study will focus on muscle growth since muscle mass is directly related to meat production. It is expected that this study will help demonstrate the potential for targeting broiler embryos to improve production in these birds. Overall, use of probiotics to improve growth and their application on eggs may serve as a novel and effective strategy for raising broilers in light of the AGP ban.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Goals / Objectives
Increasing concerns over antibiotic use in food animals and the emergence of antibiotic-resistant pathogens led to the FDA directive curbing AGP use in poultry production. Thus, there is a critical need for an effective alternative to promote poultry health and performance. Several researchers have demonstrated the potential for dietary supplementation of probiotics in day-old chicks on improving growth performance in broiler chicken. However, the period of embryonic and neonatal development accounts for almost 50% of the productive life of modern broilers and is critical to attaining quality performance at marketing. Hence, the overall goal of this study is to promote embryonic growth as a means to improve post-hatch growth in broilers.The specific objectives of this study are:1. To determine the effects of early probiotic administration on muscle growth and performance in broiler chickens.2. Identify candidate genes and pathways mediating the beneficial effects of probiotic supplementation on muscle growth in broilers.
Project Methods
Probiotic cultures: Probiotic strains (L. paracasei DUP 13076 - Lp and L. rhamnosus NRRL-B-442 - Lr) will be cultured separately in de Mann, Rogosa, Sharpe broth at 37°C for 24 h. Appropriate dilutions of the single strains in PBS will be used to obtain the desired level of inoculum (8 log CFU/egg).Experimental design, egg incubation and hatching: The study will be performed at the UConn poultry research unit. Ross 308 eggs (n=1100) will be obtained from a local commercial farm. All settable eggs will be weighed (starting egg weight), numbered and randomly assigned to the treatment groups (360 eggs/group). Group 1: Eggs sprayed with PBS (vehicle control), Group 2: Eggs sprayed with Lp, Group 3: Eggs sprayed with Lr. Eggs will be sprayed with 200 μl of the probiotic treatment strain (~8 log CFU/egg) or PBS (solvent control) on ED 0. Sprayed eggs will be incubated for 18 days at 37.5-37.8°C and 55-60% RH (Upadhyaya et al., 2015). On day 18, eggs will be transferred to the hatcher (36.8 to 37°C and 65 to 70% RH) for 3 days or until hatch. Throughout the study, eggs in different groups will be placed in separate incubators to avoid cross-contamination.Embryo morphometric measurements: Thirty-five eggs per group will be randomly sampled on ED 7, 10, 14, and 18. The eggs will be weighed and opened through the blunt end. Starting on ED 14, the embryos will be euthanized by cervical dislocation and dissected to obtain embryo weight, yolk sac weight, breast muscle weight and cross-sectional area (de Oliveira et al., 2014). Relative embryo and yolk weights will be normalized to the starting egg weight.Embryo sexing: Leg muscle sections will be collected at each sampling time for RNA extraction and qRT-PCR. Relative quantification of CHD-Z specific sequence and CHD-ZW common sequence will be normalized to the GAPDH gene. Males (ZZ) and females (ZW) will be identified based on the expression level ratio of CHD-ZW/CHD-Z. Sexual identity of the embryo will be matched back to the egg number and incorporated as metadata for statistical analysis.Hatchling morphometric measurements: On day of hatch (day 21), percent hatchability will be recorded. Thirty-five hatchlings from each group will be sacrificed and live weight, total breast muscle CSA and relative breast weight will be recorded (Aravind et al., 2003; Scheuermann et al., 2004).Broiler chicken management: Hatchlings (n=160/ group) will be sexed, weighed and tagged. Broiler chicks will be started on a 23% CP, 3000 kcal/kg ME ration and then placed on a 20% CP, 3200 kcal/kg grower/finisher ration at 3 weeks of age. Prior to feeding, individual body weights will be obtained on weeks 1-5. Feed consumed will be recorded daily on a per pen basis, uneaten food will be collected once daily before morning feeding and FCR will be calculated (Kalavathy et al., 2003).Breast weight and carcass yield percentages: On d 3, 7 (wk 1), 21 (wk 3) and 35 (wk 5) post-hatch, 35 birds from each group will be sacrificed. Head and feet will be removed, followed by defeathering and evisceration so that the carcass will be in a ready to cook (RTC) state. Dressing percentage, total breast muscle CSA, relative breast weight and relative leg weight will be recorded (Sarangi et al., 2016). Muscle collection and processing: At each sampling time [ED 10 to wk 5], breast muscle sections (n=35/embryos/birds per group) will be collected, embedded in Tissue-Tek OCT and frozen in dry ice-cooled isopentane (Al-Musawi et al., 2011; Reed et al., 2014). Additionally, samples for transcriptome analysis will be flash frozen in liquid nitrogen (Kong et al., 2017; Li et al., 2019). All samples will be stored at -80°C until further use.Muscle histology and immunostaining: Breast muscles samples (ED 10 to wk 5) embedded in OCT will be cut to 10μm thickness using a microtome cryostat and mounted to glass slides (Reed et al., 2014). Muscle sections will be immunostained using a mouse monoclonal antibody against chicken Pax7 and rabbit polyclonal antibody against laminin followed by Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG, allowing visualization of Pax7(+) satellite cells and fiber membranes. Nuclei will be detected with Hoechst 33258 (Kawakami et al., 1997; Day et al., 2009). At least 25 images will be obtained from 5 different sections of each muscle sample to quantify muscle fiber density (Scheuermann et al., 2004; Halevy et al., 2004; Reed et al., 2014; Liu et al., 2010). Fiber CSA will be measured as the region within the fiber boundary using the area measurement tool in ImageJ. Total myofiber number will be calculated by multiplying the average MFD per animal by the overall muscle CSA. The number of Pax7+ nuclei associated with the myofiber and the total number of Hoechst-stained myonuclei will be counted to determine satellite cell and total nuclei numbers (Halevy et al., 2004).Statistical analysis: A completely randomized design with factorial treatment structure will be followed. Data from morphometric measurements and muscle analysis will be sorted by age and sex and analyzed using the PROC MIXED procedure of SAS followed by Tukey post hoc test to examine statistical differences between means of different groups. Significant difference will be determined at P ≤ 0.05.RNA extraction and transcriptome sequencing: At each sampling time (ED 10 to wk 5), breast muscle sections will be harvested from 12 embryos/chicks (6 male and 6 female) per treatment group for transcriptome analysis. Briefly, RNA will be extracted from the harvested samples and their quality assessed on an Agilent 2100 Bioanalyzer (Li et al., 2019; Kong et al., 2017; Hoffman et al., 2016). For transcriptome sequencing, total cellular RNA will be rRNA-depleted, fragmented and whole transcriptome library will be constructed using the the Illumina Tru-seq paired-end (PE) RNA-seq kit. Each library will be sequenced on the Illumina NextSeq 500 (in house). We will perform PE reads of 75bp x 35bp for each library and run each on one flow lane to obtain ~80 million mappable reads (~160 million paired) per sample to ensure capture of transcripts to 0.006 copy sensitivity. We will perform biological replicates (12 eggs/hatchlings per treatment) at each time point, including validation and downstream sequencing, to strengthen statistical support for differential transcription.Transcriptome analysis: Briefly, low quality bases and duplicate reads will be removed using PRINSEQ (Schmieder and Edwards, 2011). The reads will be mapped to the chicken reference transcriptome using HISAT2 (Kim et al., 2015). Transcript abundance levels will be estimated using the IsoEM2 expectation-maximization algorithm (Nicolae et al., 2011) and differentially expressed genes will be identified using the IsoDE2 bootstrapping framework developed by Dr. Mandoiu (Mandric et al., 2017). Additionally, Database for Annotation, Visualization, and Integrated Discovery (DAVID) will be used to identify gene functional annotation terms that are significantly enriched in particular gene lists with the whole chicken genome as the background (Huang et al., 2009). DAVID will calculate a modified Fishers Exact P-value to demonstrate Gene Ontology (GO) and KEGG molecular pathway enrichment, where P-values less than 0.05 after Benjamini multiple test correction will be considered to be strongly enriched in the annotation category. Differential expression will be confirmed by RT-qPCR on a selection of 10-20 regulated transcripts (two-fold or more vs. respective control) to validate transcriptional profile across datasets.