Recipient Organization
NORTH CAROLINA A&T STATE UNIV
1601 EAST MARKET STREET
GREENSBORO,NC 27411
Performing Department
Animal Sciences
Non Technical Summary
The ruminant industry is one of the pivotal agricultural industries in the United States with a cash receipt of $67 billion, accounting for about 18% of total agricultural cash receipts in 2017. Increased demand for high-value animal-based protein is increasing the pressure on the livestock sector to meet this growing global demand. Feed cost is the major component of overall production cost in ruminant production systems. Improving the efficiency of feed utilization, therefore, can have dramatic effects on profitability and sustainability. Feed efficiency is a key criterion to improve the economic as well as environmental sustainability of the farm. Additionally, improving feed efficiency will help reduce energy losses as methane, and nitrogen as ammonia, which typically reduce animal performance and contribute to greenhouse gas emissions to the environment.The recent ban (at least in the U.S.) on the use of sub-therapeutic antimicrobials in ruminant production has necessitated a sense of urgency in the development of economically competitive alternatives such as plant nutraceuticals (essential oils, plant secondary metabolites, probiotics, prebiotics, and vaccines that do not compromise end-product quality. The major obstacle from using these alternatives is that results from different studies have been mixed. The consistent results with the use of sub-therapeutic antimicrobials are lacking in these alternatives. Some of the factors responsible for the mixed results with the alternatives are diet composition, dosage, type of animal, production level, enzyme activity, rumen condition, adaptation, additives-microbiome interactions, etc.The use of plant nutraceuticals in livestock production continues to get traction despite the mixed results (short-term effect and inconsistent effect across-the-board) associated with their use. To build on previous studies, the team will use the proposed project to develop a product mix to improve gut health and feed efficiency in ruminants. The product blend (or mix) will be a combination of different products that the PD has used to improve feed efficiency and animal productivity. There is knowledge gap on long-term in vivo evaluation of these nutraceutical plants. The proposed project will evaluate the long-term effects of previously studied nutraceuticals in order to overcome the adaptation of the rumen microbiota to nutraceuticals. Data generated will contribute to decreasing the knowledge gap in these nutraceuticals and we plan to develop a product mix that will consistently work across-the-board. We hypothesize that combining these nutraceuticals will have synergistic effects on gut health and feed efficiency. Rumen and blood samples will be used for cytokines, microbiome and metabolome analyses generating big data which will provide additional insight into solutions to improve gut health and feed efficiency in ruminants.
Animal Health Component
60%
Research Effort Categories
Basic
10%
Applied
60%
Developmental
30%
Goals / Objectives
Increased demand for high-quality animal-based protein is increasing the pressure on the livestock sector to meet this growing global demand which is largely driven by increasing human population. To meet this huge demand, the livestock industry has used an assortment of feed additives to improve feed efficiency and nutrient utilization, promote growth, enhance gut health and animal welfare, and abatement of greenhouse gas emissions. The predominant practice was the administration of antibiotics at low doses (sub-therapeutic use) which consistently increased performance parameters (growth, reproduction and health) across-the-board. But this practice has been discontinued due to scientific and public concerns regarding the risk of antibiotics resistance to humans.The recent ban (at least in the U.S.) on the use of sub-therapeutic antimicrobials in ruminant production has necessitated a sense of urgency in the development of economically competitive alternatives such as plant nutraceuticals (essential oils, plant secondary metabolites, probiotics, prebiotics, and vaccines that do not compromise end-product quality. The major obstacle from using these alternatives is that results from different studies have been mixed. The consistent results with the use of sub-therapeutic antimicrobials are lacking in these alternatives. Some of the factors responsible for the mixed results with the alternatives are diet composition, dosage, type of animal, production level, enzyme activity, rumen condition, adaptation, additives-microbiome interactions, etc.Despite not having any product(s) as effective as antibiotics, the global plant nutraceutical industry continues to grow and is predicted to reach $111 billion by the end of 2023. The increase in the demand and use of plant nutraceuticals is attributed to the increasing consumer and public preferences towards wholesome meat production (preference is tilted towards no synthetic antibiotics and growth promoters use). Nutraceutical plants contain some biologically active compounds, generally phenolic-based compounds, with an ability to alter ruminal fermentation and change ruminal microflora community. Polyphenolics and essential oils are the main active components of most nutraceuticals. As stated earlier, several studies have documented the benefits of plant nutraceuticals use in livestock production but inconsistent results and short-term effect of some of these nutraceuticals need to be addressed. The short-term effect of some of these nutraceuticals in improving production and health metrics is mainly caused by adaptation of the rumen microbiome to the nutraceuticals. Nutraceuticals whose effects in improving feed efficiency and gut health (and reducing greenhouse gas emissions) are limited to a short period of time (30 days or less) are not suitable as long-term solution in ruminants. Using a blend or mixture of some promising plant nutraceuticals may be a sensible approach towards addressing the limitations associated with their use in livestock production. This is not an entire new concept as the industry already combine probiotics and prebiotics to form synbiotics. And studies have shown that synbiotics exhibited synergistic effects such as increasing the proliferation of beneficial bacteria and boosting the perpetuation of newly introduced probiotics in the host; with up to a 100-fold increase in intestinal bifido-bacteria population with the introduction of prebiotics. Additionally, a combination of a commercially available prebiotic and probiotic has been shown to eliminate morbidity and mortality losses associated with Shiga toxin-producing Escherichia coli infections in dairy calves. There are documented reports on combining probiotics and enzymes and a blend of essential oils in literature but there is a significant gap on the effect of combining different essential oils and polyphenolics.The novel idea in the proposed project is the mixing of different plant nutraceuticals that the principal investigator has evaluated previously with some levels of success. The rationale is that the effects of the nutraceutical blend will not be limited to short period of time due to adaptation of the rumen microbiome. The team will combine 9 previously researched plant nutraceuticals to formulate a blend that will have synergistic (positive) effects to increase feed efficiency and improve gut health in ruminants. To ensure that results generated can be interpreted biologically, the team will be using microbiome, metabolome and cytokine analyses (big data analytics) to validate animal productivity measurements. The big data analytics will provide additional insight into what is driving the observed changes in feed efficiency and gut health.The broad objective of the proposal is to develop a feed additive blend from 9 plant nutraceuticals (Moringa oleifera, Corymbia citriodora, Ocimum gratissimum,, Juniperus communis, brown and red onion peels, lemon grass oil, rosemary oil and algae oil) to manipulate ruminal fermentation, improve feed efficiency and animal productivity, and enhance gut health in ruminants. The proposed project aims to build on previous studies on these nutraceuticals by the team. The team will evaluate all possible combinations and ratios for these 9 nutraceuticals as synergistic and additive interactions depending on the combined ratio. We hypothesize that combining different plant nutraceuticals will enable the team to develop a superior product blend that will improve feed efficiency and gut health in ruminants. This will be addressed by following these specific objectives:1) Extract and estimate the bioactive components of 9 different plant nutraceuticals;2) Use the in vitro batch culture technique to screen different combinations of the nutraceuticals to improve feed efficiency;3) Use the rumen stimulating technique (RUSITEC) to further evaluate the different product blends;4) Evaluate the effects of three final product blends on feed intake, nutrient digestibility, rumen fermentation, and milk yield and composition of dairy cows;5) Evaluate their effects on the microbiome, metabolites and cytokines; and6) Develop a final product blend that will consistently improve feed efficiency and gut health in ruminants.
Project Methods
Institutional Animal Care and Use Committee approval will be obtained for all studies involving animals.Objective 1: Collect, extract and estimate the bioactive components of different nutraceutical plants and unsaturated oilsNine nutraceuticals (M. oleifera, C. citriodora, O. gratissimum,, J. communis, brown and red onion peels, lemon grass oil, rosemary oil and algae oil) will be utilized for this project. Concentration of total tannins in the extracts will be determined according to Makkar (2003). Saponins will be determined according to Ahmed and Wang (2015) and total phenolic content will be determined chromatographically as described by Makkar (2003). The composition of the essential oils will be determined by using a gas chromatography-mass spectrometry (GC-MC) according to Cobellis et al. (2016)Objective 2: Use the batch culture technique to screen different combinations of the nutraceuticals to improve feed efficiencyThe in vitro batch culture technique will be used to screen the 9 plant nutraceuticals according to Anele et al. (2015; JAS 93, 4852-4859). The study will be a factorial design with 2 (substrates) × 81 (or more nutraceutical combinations) × 3 (doses) + control arrangement. There will be positive (a commercial feed additive) and negative (no feed additive) control treatments. Substrates will be high forage and high concentrate rations. Dose of nutraceuticals will be low, medium and high concentrations. Gas production will be measured at 3, 6, 9, 12, and 24 h. Dry matter disappearance will be determined after 3, 6 and 24 h and the residue will be analyzed to determine NDF and ADF disappearance. Gas measurements will also include in vitro methane and ammonia concentrations. Additionally, VFA and efficiency of microbial production will be determined. Any product blend that increases DM digestibility, VFA and efficiency of microbial production by at least 10% with concomitant decrease in methane and ammonia concentrations will be selected and further evaluated using a RUSITEC. The 10% selection baseline will be increased to 20% or more if more than 30 of the product blends meet this criterion.Objective 3: Use the rumen stimulating technique (RUSITEC) to further evaluate the different product blendsThe RUSITEC that will used in the study has six 800 ml capacity fermenters and the general incubation procedure will be as described by Czerkawski and Breckenridge (1977). Three product blends will be duplicated and evaluated in each run. The experiment will be a duplicated 3 × 3 Latin Square design with 3 periods of 15 days. In each period, there will be a 10-day washout period followed by 5 days of sampling and data collection.Dry matter disappearance will be determined after 48 h. Residues will be ground through a 1-mm screen for chemical analysis. Fermenter pH and liquid effluent volume will be recorded daily and sub samples taken to analyze for ammonia (Rhine et al., 1998) and VFA (Anele et al., 2014) concentrations. Utilizable crude protein at the duodenum will be estimated as per the procedure of Lebzien and Voigt (1999). Based on the results, three product blends will be selected for final evaluation.Objective 4: Evaluate the effects of three product blends on feed intake, nutrient digestibility, rumen fermentation and milk yield and composition of dairy cowsIn vivo studies will be carried out to determine the effects of selected product blends on feed utilization and lactational performance of dairy cows. Eight cannulated Holstein cows will be randomly assigned to three treatments (selected based on the RUSITEC study) and a control treatment (a commercial feed additive) in a duplicated 4 ´ 4 Latin square design, with four periods of 21 days. The cows will be offered a total mixed ration, top-dressed with each treatment. The total mixed ration consists of corn silage (250) and a concentrate mix (750). Feed, orts and fecal grab samples will be collected on d 15 through 21 and composited for each cow in each period. To determine digestibility, acid insoluble ash will be used as the internal marker. Ruminal fluid samples will be collected from day 15 to 21 of the study to determine ruminal pH, VFA and lactic acid concentrations. Rumen contents will be strained through 4 layers of cheesecloth and the pH of ruminal fluid will be measured immediately with a digital pH meter. A subsample of rumen fluid will be used for the analysis of ruminal ammonia-N and VFA. Daily milk samples will be collected from all the lactating cows throughout the study - 84 days, and analyzed for SCC, MF, SNF, protein, lactose, TS and pH.Objective 5: Evaluate the effects of the product blends on the microbiome, metabolome and cytokines of dairy cows DNA ExtractionSamples of rumen digesta will be freeze-dried and thoroughly mixed by physical disruption using a bead beater (1 min at 5,000 rpm) (Mini-bead beater 8, BioSpec Products, Bartlesville, United States). The extraction of total DNA will be performed from 50 mg samples using the QIAampR DNA StoolMini Kit (Qiagen USA) following the manufacturer's instructions.Amplification of the V1-V2 hyper-variable regions of 16S rRNA will be carried out with primers 27F and 357R (Liu et al., 2007). The protocol that will be followed for PCR amplification, short fragment removal and sequencing has been described by Martínez-Fernández et al. (2015). After amplification, individual libraries will be cleaned, and quality control done to determine library size and DNA concentration. Libraries will then be normalized and pooled for Next Generation Sequencing (NGS). The Illumina MiSeq (600 cycles) (V3) flow cell: PE 300 protocol and sequencing will be completed at Georgia Genomics and Bioinformatic Core facility of University of Georgia, Athens.To estimate health benefits of the treatments, blood samples will also be collected on day 0, 15 and 21 for non-targeted immune related gene expression (about 80 genes). Neutrophils will be extracted from the blood and used for this analysis. Reason - Neutrophils are the most abundant leukocytes in humans and animals and among the first cells to arrive at the site of inflammatory immune response (Oh et al., 2008). Isolating RNA from neutrophils captures more of the message generated at the time of blood collection (Oh et al., 2008).Non-Targeted Metabolomics AnalysisRumen fluid samples will be prepared and analyzed according to the procedure of Want et al. (2013) with modification by Ogunade et al. (2018). This analysis will generate about 160 (139 ± 21) metabolites.Objective 6: Develop a final product blend that will consistently improve feed efficiency, milk yield and composition, and gut health in ruminantsFor this study, 20 multiparous lactating Holstein cows will be stratified by day in milk, milk production and somatic cell counts and randomly assigned to one of five treatments consisting of three product blends from the Latin square study (objective 4), a positive control (a commercial feed additive) and a negative control (no feed additive). Treatment will be top-dressed into their ration and the study will be for 60 days.Cows will be maintained as described previously. Feed, orts, fecal and milk samples will be analyzed as previously described. Milk efficiency will be estimated as milk yield/DM intake (DMI) and energy corrected milk/DMI.Blood samples will be collected on day 0, 15, 30, 45 and 60 for non-targeted immune related gene expression (about 80 genes) and non-targeted metabolome analysis (about 160 metabolites) as described in objective 5.At the end of the study, the product blends will be ranked based on their effects on production parameters and gut health, and these ranking must be validated by results from the immune related gene expression and metabolome analyses.