Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
(N/A)
Non Technical Summary
Improving broiler meat production to meet consumer demand has resulted in the shortening of the time required for modern broilers to reach market weight. Due to this, the period of embryonic development constitutes to about 30% of a broiler's life. Further, optimum growth during the embryonic period and immediately prior to hatch is important to chick performance in reaching market weight. Hence supporting this critical embryonic period is critical to promote overall bird health and production. Along these lines, probiotics are one of the most common growth promoters employed in broiler production. However, they are primarily used as a feed supplement post-hatch and their effect on embryonic growth has not been studied. Given their beneficial attributes, supplementation of probiotics to growing embryos could be a potential approach to promote growth and performance in broilers. Towards this, our previous research demonstrated that providing probiotics to growing embryos not improved embryo growth but also promoted body weight gain and meat yield in chickens. Further, we also observed that probiotics also improved hatchability and hatchling quality. One of the key determinants is the energy status of the embryos. Additionally, the improvement in growth can be due to improved intestinal function including cecal microbial activity. Therefore, to better understand how probiotics promote growth, in this project we will determine probiotic effect on the glucose and glycogen reserves, intestinal development and function and cecal microbiome in the embryo and chicks. 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.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Over the last few decades, there has been an increasing demand for poultry meat as a source of high-quality protein for human consumption. To meet this demand, the poultry industry employed planned genetic selection targeting higher feed efficiency and performance. In addition to maximizing body weight, there has been a considerable shortening in the growing period. In effect, in modern broilers, the period of embryonic and neonatal development represents almost half of its productive lifespan. Particularly, the perinatal and immediate postnatal period is a crucial time in the development of the young chick influencing quality broiler performance at marketing. Consequently, any approach that supports embryonic and neonatal growth is expected to have a significant effect on overall health and performance of broiler chicken. In this regard, probiotics are widely used as growth promoters in the poultry industry. However, probiotic application is limited to in-feed supplementation post-hatch and their effect on embryonic growth has not been studied. Moreover, poultry researchers have now realized that future gains in production potential of these birds will come from advancements made on embryogenesis during incubation. Therefore, given their beneficial attributes, in-ovo supplementation of probiotics, could be a potential and viable approach to support perinatal and neonatal growth thereby enabling the chicken to better express its genetic potential. Hence, the overall goal of this study is to elucidate the effect of in-ovo probiotic supplementation on the energy status, intestinal development and microbiome acquisition in the growing embryo and chicksThe specific objectives of this study are:1. To determine the effect of in-ovo probiotic supplementation on energy status of perinatal broiler embryos, hatchlings and chicks2. To determine the effect of in-ovo probiotic supplementation on the structure and function of the yolk sac membrane (YSM) and small intestine (jejunum) in broiler embryos and chicks3. Identify the effect of in-ovo probiotic supplementation on the composition and diversity of the gut (cecal) microbiome of the growing embryo and broiler chicks.
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.Objective 1: To determine the effect of in-ovo probiotic supplementation on energy status of perinatal broiler embryos, hatchlings and chicksEmbryo morphometric measurements: Twenty eggs per group will be randomly sampled on ED 14, 18, 19 and 20. The eggs will be weighed and opened through the blunt end. The embryos will be euthanized by cervical dislocation and dissected to obtain embryo, yolk sac, breast muscle and liver weight. Relative embryo and yolk weights will be normalized to the starting egg weight.Hatchling morphometric measurements: On day of hatch (day 21), percent hatchability will be recorded. Twenty hatchlings from each group will be sacrificed and live weight, residual yolk sac, breast muscle and liver weight will be recorded.Broiler chicken management: Hatchlings (n=60/ group) will be sexed, weighed and tagged. Broiler chicks will be fed on a 23% CP, 3000 kcal/kg ME ration for three weeks. Prior to feeding, individual body weights will be obtained on weeks 1and 3. 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. On week 1 and 3 post-hatch, 20 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 and liver weight will be recorded.Tissue sampling: Yolk samples collected on day 0 will be weighed, homogenized and stored at -20°C prior to analysis. Further, at each sampling day (d14, 18, 19, 20 and 21) YS contents will be separated from the YSM, weighed, homogenized and stored at -20°C until further analysis (Yadgary and Uni, 2012). In addition, liver and pectoral muscle samples will be collected at each sampling time (d14, 18, 19, 20, 21, week 1 and 3 post-hatch), frozen in liquid nitrogen and stored at -80°C for glucose and glycogen analysis (Uni et al., 2005).Objective 2. To determine the effect of in-ovo probiotic supplementation on the structure and function of the yolk sac membrane (YSM) and small intestine (jejunum) in broiler embryos and chicks.Morphometry and tissue sample collection: At each sampling time, 20 embryos per group will be randomly selected and euthanized by cervical dislocation. Embryo weight, intestinal weight and residual yolk sac weight will be recorded. Similarly, morphometric measurements including body weight, yolk sac weight, intestinal weight and feed consumption will be recorded for the hatchlings and chicks. For YSM, tissue samples will be collected from 10 embryos/group on d13, 15, 17, 19 and 21. Similarly, for the intestine, jejunal sections will be collected from 10 embryos/chicks at different times [Embryo: 15, 17, 19, 20, 21(day of hatch) and chick: day 3, 7 and 14 post-hatch] for histology. Additionally, eight embryos/chicks from each treatment will be sampled at each sampling time for YSM and intestinal (jejunum) transcriptome analysis.Yolk Utilization and Yolk Sac Histology: The embryo weight and residual yolk sac weight will be recorded and residual yolk percent relative to embryo weight will be calculated. YSM will be dissected from the area vasculosa and fixed overnight in 4% buffered formaldehyde. The perimeters of the endodermal epithelial cells (EECs; 10-15 cells) in the YSM sections from each embryo will be measured to assess size of the absorptive EECs.Jejunal morphometry: The central part of the jejunal segment will be fixed in 4% paraformaldehyde for histology and immunohistochemistry. The variables assessed will include the following: villus length, villus diameter, depth of crypts, goblet cell diameter, crypt goblet cell number and villus goblet cells type number (acidic, neutral or mixed).RNA extraction and transcriptome sequencing: At each sampling time, YSM and Jejunal samples will be processed for RNA extraction, library generation and transcriptome sequencing using standard protocols.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. Transcript abundance levels will be estimated using the IsoEM2 expectation-maximization algorithm and differentially expressed genes will be identified using the IsoDE2 bootstrapping framework. 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.Objective 3: Identify the effect of in-ovo probiotic supplementation on the composition and diversity of the gut (cecal) microbiome of the growing embryo and broiler chicks.Sampling , Microbiome Sequencing and Data Anlysis: At each sampling time [d 15, 17, 19, 21(day of hatch), and d 3, 7, 10 and 21 post-hatch], eight eggs/birds from each treatment and control group will be sampled for microbiome analysis. Cecal microbial diversity in the embryo and chicks will be determined by shot-gun sequencing using the Illumina platform. The 16S rRNA gene sequence analysis will be performed using QIIME2. The sequence reads will be demultiplexed by per-sample barcodes, and DADA2 used to correct the Illumina-sequenced amplicon read errors. Sequences from a phylogenetic tree will be taxonomically classified using the Greengenes reference database at a confidence threshold of 99%. The α and β diversity will be calculated using the Shannon Index and the Jaccard similarity distance matrix, respectively. Linear discriminant analysis effect size (LEfSe) will be conducted according to the relative abundances of each bacterial community to verify the highlights that significantly differed between the samples. Metagenome functional predictive analysis of the communities will be performed via phylogenetic investigation in PICRUSt software.