Performing Department
(N/A)
Non Technical Summary
In modern broilers, the first week post-hatch is associated with rapid muscle growth and represents almost 20-25% of the total production period. Hence, any nutrient and energy limitations during this critical phase, such as experienced during post-hatch feed deprivation, can limit muscle growth and overall productivity. Towards this, early nutritional supplementation via in-ovo feeding was identified as a potential approach to jump-start development in chicks. Along these lines, although widely used as a growth promoter, probiotics have not been evaluated as an in-ovo intervention to minimize the deficiencies caused by delayed feed access. In this regard, data from a synergistic USDA grant demonstrate that in-ovo spray application of probiotics promoted embryonic growth, energy reserves in the perinatal embryo and post-hatch performance. Further, preliminary observations indicate a role for probiotics in promoting muscle development including satellite cell activity, myofiber hypertrophy and muscle growth in chicks subject to delayed feed access. However, this warrants further investigation. Therefore, as a next step, the proposed research aims to determine the effects of in-ovo probiotic supplementation on mucle development in chicks subject to post-hatch feed deprivation and elucidate the underlying molecular mechanisms. Ultimately, we expect that in-ovo probiotic application can be integrated with routine management practices to achieve optimal muscle growth and subsequent meat production in broilers, thereby promoting the poultry industry.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Goals / Objectives
In broiler chicken, the breast muscle is the largest muscle by weight and sought out as a lean meat choice. In fact, it is the most valuable portion of the chicken carcass. Therefore, even small differences in breast yield can have a significant economic impact. Consequently, the productivity of a food animal is directly related to the cellular and molecular mechanisms regulating skeletal muscle myoblast growth and development, particularly in the pectoral muscle. Towards this, the first few days post-hatch represent a critical phase in skeletal muscle development in broilers. Thus, any imbalance in nutrient and energy needs at this critical phase, such as experienced during post-hatch feed deprivation, may limit maximal growth of the chicks thereby impeding subsequent performance. In this regard, early nutritional programming via in-ovo feeding has been identified as a potential approach to jump-start development in chicks. Towards this, our preliminary data demonstrate that in-ovo probiotic application supported muscle development in chicks subject to delayed feed access by significantly improving satellite cell activity, myofiber maturation and muscle mass accretion through modulation of key myogenic factors. Further, we observed an improved energy status in these birds via increased glycogen reserves. However, this warrants further investigation. Therefore, in this application we aim to determine the extended effects of in-ovo probiotic supplementation on muscle growth and overall productivity during the grow-out period and characterize the underlying molecular mechanisms. Understanding how in-ovo probiotic supplementation supports muscle growth and development will help identify key physiological processes and factors that help reduce the deficiencies caused by delayed feed access. Ultimately, we expect that that in-ovo probiotic spray application could be potentially employed to achieve optimal muscle growth and subsequent meat production in broilers, thereby promoting the poultry industry, a key agricultural sector supported by the USDA.The specific objectives are:To determine the effects of in-ovo probiotic supplementation on glycogen reserves, productivity and meat quality in birds subject to post-hatch feed deprivation.To determine the effects of in-ovo probiotic supplementation on muscle growth and development in birds subject to post-hatch feed deprivation.Characterizing the dynamic changes in pectoral muscle proteome as influenced by in-ovo probiotic supplementation in birds subject to post-hatch feed deprivation.
Project Methods
Obj.1: To determine the effects of in-ovo probiotic supplementation on glycogen reserves, productivity and meat quality in birds subject to post-hatch feed deprivationProbiotic culture: 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, in-ovo treatment and hatching: Ross 308 eggs (n=1080) will be weighed and randomly assigned to one of four treatment groups namely i) Control-48: Eggs sprayed with PBS (vehicle control) and hatchlings subject to 48 h feed deprivation following hatch, ii) Contol-72: Eggs sprayed with PBS (vehicle control) and hatchlings subject to 72 h feed deprivation following hatch iii) IO-48: Eggs sprayed with LP and hatchlings subject to 48 h feed deprivation following hatch and iv) IO-72: Eggs sprayed with LP and hatchlings subject to 72 h feed deprivation following hatch. Sprayed eggs will be incubated for 18 days at 37.5-37.8°C and 55-60% RH. 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.Broiler chicken management: Hatchlings (n=120/group) in each group will be weighed and randomly assigned to four replicate pens. Following feed deprivation period, 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 starting at 3 weeks of age until the end of the study (6 weeks). 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).Morphometric measurements: At each sampling time, 12 birds per group (3 birds/pen) will be sacrificed and live weight, residual yolk sac, breast muscle, liver, spleen, pancreas, gizzard, proventriculus and small intestine (duodenum, jejunum, ileum) weight will be recorded.Tissue sampling: At each sampling day (d0, 1, 3 and 5), yolk sac contents will be separated from the yolk sac membrane and stored at -20°C until further analysis. In addition, liver and pectoral muscle samples will be collected at each sampling time (d0, 1, 3, 7, 14, 28 and 42), frozen in liquid nitrogen and stored at -80°C for glycogen, protein and triglyceride analysis (Uni et al., 2005; Wang et al., 2020; Bhanja et al., 2009).Glycogen, protein and triglyceride analysis: Samples of homogenized yolk sac contents, liver and pectoral muscle will be processed as previously described. Similarly, total protein and triglyceride content will be estimated using standard protocols (Wang et al., 2020; Bhanja et al., 2009).Meat quality: At the end of the study period (day 42), breast muscle from 12 birds/group will be subject to meat quality analysis including meat color (CIE L*, a* and b*), drip loss, lipid oxidation, myofibrillar fragmentation, pH and Warner-Bratzler shear force using standard protocols.Obj.2: To determine the effects of in-ovo probiotic supplementation on muscle growth and development in birds subject to post-hatch feed deprivationExperimental design, egg incubation and hatching: In each trial, we will include a total of 600 eggs, with the eggs randomly assigned to one of four groups (150 eggs/group) as in objective 1. Probiotic culture preparation, in-ovo application, experimental groups, incubation and hatching will be performed as described in objective 1. On hatch, male chicks in each group will be weighed, transferred to floor pens (30 birds/pen; three pens/group), subject to feed deprivation and managed as previously described.Sampling: On day 0 (at hatch), 1, 2, 3, 5, 7, 10, 28 and 42, six birds per group (two birds/pen) will be sacrificed and body weight and breast weight will be recorded. These sampling times are based on significant differences observed in muscle histology, satellite cell activity and muscle morphology (Kornasio et al., 2011; Velleman et al., 2014a,b; Powell et al., 2016a,b).Pectoral muscle sample collection and processing: At each sampling time, pectoral muscle samples (upper right pectoralis major muscle) for histology will be embedded in Tissue-Tek OCT and frozen in dry ice-cooled isopentane. Muscle samples (upper left pectoralis major muscle) for proteomic analysis will be collected and flash frozen in liquid nitrogen (Zhang et al., 2021). All samples will be stored at -80°C until further use.Muscle histology and immunostaining: Pectoral muscles samples embedded in OCT will be cut to 10μm thickness using a microtome cryostat, mounted to glass slides and immunostained for chicken Pax7 (Reed et al., 2014; Kawakami et al., 1997). Myofiber cross section area, myofiber and nuclear density will be determined. The number of Pax7+ nuclei associated with the myofiber along with the total number of Hoechst-stained myonuclei will be counted to determine satellite cell and total nuclei numbers (Halevy et al., 2004).Muscle morphology: Muscle sections will be cut, mounted to glass slides and stained with hematoxylin and eosin as previously described (Kornasio et al., 2011; Powell et al., 2016 a,b). One stained muscle section per bird will be imaged and spacing width between 10 separate pairs of adjacent fiber bundles will be measured. The images will also be scored by four trained individuals for muscle morphology as described in Velleman et al. (2003). Additionally, imaged sections will be scored for their adiposity by four trained observers with scores ranging from 1 to 5. (Powell et al., 2016b; Velleman et al., 2014b).Myonuclear apoptosis assay: We will use the standard TUNEL assay to quantify apoptotic myonuclei. Briefly, pectoralis major muscle sections will be cut, stained for double-stranded DNA breaks using TUNEL labeling, and counter stained with propidium iodide. The sections will be imaged, and the index of apoptotic labeling will be calculated by expressing the number of fluorescein-labeled nuclei relative to propidium iodide-labeled nuclei as previously described (Mozdiak et al., 2002; Pophal et al., 2003).Satellite cell proliferation and differentiation assay: Satellite cells from pectoral muscle samples will be harvested and cultured as previously described (Flees et al., 2022; Harding et al., 2016). Satellite cell proliferation will be evaluated using BrdU incorporation assay (Day et al., 2009). The ability of satellite cells to differentiate will be measured by fusion index.Obj 3: Characterizing the dynamic changes in pectoral muscle proteome as influenced by in-ovo probiotic supplementation in birds subject to post-hatch feed deprivationTissue sampling, sample prep and proteomic analysis: At each sampling time (day 0, 1, 3, 7, 28, 42), pectoralis major muscle sample will be collected from six birds in each group and stored as described under objective 2. Sample prep and proteomic analysis will be performed in collaboration with the UConn Proteomics and Metabolomics Facility.Bioinformatics and statistical methods: Raw MS/MS data will be processed with MaxQuant software (v. 1.6.10.43) and searched against the Uniprot Gallus gallus proteome (UP000000539) using selected variable modifications and a fixed carbamidomethyl Cys modification. Peptide and protein quantification will be achieved using MaxQuant's LFQ algorithm (Cox and Mann, 2008). After total ion intensity normalization, the spectrum count/abundance of identified proteins/peptides will be further analyzed using the limma package in R (Zhai et al., 2020; Dang et al., 2022; Johnson et al., 2023). Log2 fold change threshold will be applied for differentially abundant protein identification. Detailed protein functions will be annotated based on Uniprot database (2021) with verification through literature search.