Performing Department
Animal Science
Non Technical Summary
Increasing concerns over antibiotic use in animals and the emergence of antibiotic resistant pathogens resulted in the FDA directive curbing use of antibiotic growth promoters (AGPs) in food animals including poultry. This has led to an urgent need for safe and natural alternatives to AGPs in promoting poultry health and performance. In this regard, several researchers have demonstrated the efficacy of probiotic supplementation to day-old chicks in improving performance in market birds. However, the period of embryonic growth and immediate post-hatch development account for almost half of the productive life of modern broilers. Furthermore, this developmental period is critical to attaining quality broiler performance at marketing. Therefore, early probiotic administration during embryonic development could be a potential and viable approach to promote sustained growth and development in broilers. Hence, this study will investigate the effect of probiotic administration during early embryonic development on promoting growth in embryos, hatchlings and broiler chicks. Ultimately, successful completion of this research can be expected to provide a feasible and practical treatment that can be applied in conjunction with routine poultry management practices to promote health and performance in chickens.Our central hypothesis is that probiotics significantly enhance embryonic growth and performance in broiler chicken by modulating the intestinal microflora and promoting intestinal function. We have based our hypothesis on published reports and our own preliminary studies, in which we demonstrate that in-ovo and in-feed application of probiotic cultures significantly improved growth and feed conversion ratio in layer embryos and pullets. Further, these changes were found to be associated with a significant increase in cecal lactobacilli population in probiotic treated chicks. Additionally, similar studies conducted with broiler lines (Ross 308) demonstrated a significant increase in body weight, morphometric measurements and cecal microbial population in embryos and hatchlings. The specific objectives of this study include:i) To determine the effect of in-ovo and in-feed probiotic administration on embryonic growth and post-hatch performance in broiler chickensii) To identify the effect of probiotic supplementation on the composition and diversity of the gut microbiome in the growing embryo, hatchling and broiler chicken.iii) To identify the effects of probiotic supplementation on intestinal function in the growing embryo, hatchling and broiler chicken.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The goals of the proposed study are to investigate the effect of early (in-ovo) and sustained (in-feed) probiotic supplementation on promoting growth and performance in broilers. Additionally, the effect of probiotic supplementation on the chicken gut microbiome and intestinal function will be investigated.
Project Methods
Objective 1. Experimental design: All animal experiments will be conducted with the approval of the UConn Institutional Animal Care and Use Committee. Freshly laid fertile eggs (Ross 308) will be obtained from a local commercial farm. A total of 1980 eggs will be needed for the study, with 660 eggs for each of the three replications. Eggs in each trial will be randomly assigned to one of the four experimental groups. The treatment groups include: i) Control (no probiotic treatment), ii) Lactobacillus paracasei DUP-13076 (LP), iii) L. rhamnosus NRRL-B-442 (LR) and iv) Probiotic cocktail (PC). Based on the power analysis (Lenth, 2007), we will sample 12 eggs/birds per treatment/time point to obtain a significant difference in morphometric and performance parameters between treated and control eggs/birds.Probiotic culture conditions and preparation of probiotic spray: Probiotic strains (Lactobacillus paracasei DUP 13076 and L. rhamnosus NRRL B-442) were selected based on our previous studies and preliminary layer trial (Muyyarikkandy and Amalaradjou, 2017). Each strain will be cultured separately and washed twice with sterile PBS. Equal portions from each of the two strains will be combined to make the two-strain probiotic cocktail. Appropriate dilutions of the cultures in PBS will be used to obtain the desired level of inoculum (9 log CFU/egg).Egg treatment, incubation and hatching: Freshly laid eggs will be transferred to the avian research facility at UConn. All settable eggs will be weighed individually, numbered and randomly assigned to the different treatments group. On day 0, 3, 7, 10, 14 and 18 of incubation, eggs will be sprayed with 200 µl of PBS (group 1) or probiotic (9 log CFU/egg in groups 2-4). The sprayed eggs will be incubated in a thermostat incubator for 18 days under standard conditions (Upadhyaya et al., 2015). On day 18, the eggs will be removed, weighed individually followed by probiotic/PBS treatment application and transferred to the incubator set up for hatching for 3 days or until hatch.Embryo sampling and morphometric measurements: Twelve eggs per treatment will be randomly sampled on days 0, 7, 10, 14 and 18 of incubation. The eggs will be weighed and opened through the blunt end for morphometric assessments (O'Dea et al., 2006; de Oliviera et al., 2014). The embryos will be euthanized by cervical dislocation prior to dissection. Embryonic development will be evaluated by the absolute and relative weights of body, yolk and organs as described by Li et al. (2013). Briefly, the thorax, leg, heart, liver, stomach and intestines will be excised and weighed. Additionally, starting on day 14, jejunum and its contents will be collected for further analysis in objective 2 and 3.Hatch sampling: On day of hatch (day 21), percent hatchability will be recorded and hatchlings will be weighed prior to placement on floor pens. Twelve hatchlings from each treatment group will be sacrificed and morphometric measurements will be performed as described under embryo sampling. Organ weights will be expressed relative to body weight (Aravind et al., 2003). Additionally, jejunum and its contents will be collected for further analysis in objective 2 and 3.Broiler chicken, diet and management: Hatchlings will be weighed and tagged. They will be housed on floor pens with wood shavings as bedding and supplemental heat at the UConn avian facility. Broiler birds 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. The feed will be supplemented daily with 200 µl of PBS or probiotic (9 log CFU/g of feed) from hatch until sacrifice (week 5).Body weight and Feed conversion ratio: Prior to feeding, individual body weights will be obtained on weeks 1-5. Feed consumed will be recorded daily on per pen basis, the uneaten food will be collected once daily before morning feeding and feed conversion ratio will be calculated (Kalavathy et al., 2003).Organ weights, abdominal fat deposition and carcass yield percentages: On weeks 1-5, twelve birds from each treatment group will be sacrificed. Head and feet of the bird will be removed, followed by defeathering, and evisceration. Carcass, breast and leg weight will be measured and expressed as a percentage of live body weight (Sarangi et al., 2016). Internal organs including gizzard, heart, liver, spleen, small intestine, cecum, and colon will be collected. Both absolute and relative percent of organ weight to the body weight will be calculated (Kalavathy et al., 2003). Additionally, jejunum and its contents will be collected for further analysis in objective 2 and 3.Objective 2. Microbiome sequencing: Briefly, at each sampling time (d14, d18, d21, wk1, wk3, and wk5), jejunal contents (n=6/group) will be collected and stored at -80ºC until further processing. Sequencing will be performed at the MARS Facility at UConn. DNA from the jejunal contents will be extracted and DNA libraries will be constructed according to the Illumina MiSeq DNA Sample Prep Guide. DNA from the samples will be PCR amplified using primers targeting the V4 region of the bacterial 16S rRNA gene (V4). Sequencing will be performed on the Illumina MiSeq2000 platform according to the manufacturer's protocol (Hsieh et al., 2017).Objective 3. Sample collection: At each sampling time (d14, d18, d21, wk1, wk3, and wk5) under objective 1, jejunum (n=6/group) will be excised and segments (1 cm long) will be placed in three separate tubes: 1) fixed in buffered formalin for histology, 2) collected in RNAlater and stored at -80°C for qPCR and 3) stored at -20°C for determination of enzyme activity (Uni et al., 2003).Histomorphological analysis: Since the height of villus in the ileum indicates the turnover of epithelium and thereby nutrient metabolism, the villus height will be measured in this study (Demir et al., 2003; Awad et al., 2009). Briefly, formalin fixed samples will be processed to measure villus height and crypt depth as described by Awad et al (2009). Further, villus surface area will be calculated from villus height and width at half height (Tako et al., 2004).Real-time qPCR: For the assay, RNA will be extracted from the jejunal sections using RNeasy Plus kits (Qiagen), subject to cDNA synthesis and RT-qPCR will be performed using SYBR green chemistry (Invitrogen). Primers for genes encoding digestive enzymes (Aminopeptidase, isomaltase, glucoamylase) and transporters (Oligopeptide transporter, glucose and galactose transporter and Sodium-potassium transporter) will be evaluated for their differential gene regulation with reference to gapdh expression (endogenous control; Bages et al., 2015; Fernandez-Alarcon et al., 2017). The comparative Ct method (2-ΔΔCt) will be used to assess differential gene expression (Bookout and Mangelsdorf, 2003).Brush border enzyme activity: Enzyme activities will be assayed in homogenized jejunal tissue. Maltase activity will be colorimetrically assayed using maltose as a substrate and aminopeptidase activity will be determined by hydrolysis of p-nitroanilide (Uni et al 2003; Murugesan et al., 2014).