Progress report for GNE24-328
Project Information
Egg production is an important animal industry in the Northeast region such as Pennsylvania and New York. However, egg farmers, particularly small-scale local farmers, often face challenges including marginal profits, avian influenza outbreaks, and disrupted supply chain. Feeding hempseed meal and microalgae in laying hens to produce health-promoting and low-carbon eggs is one innovative strategy to improve the economic resilience and
social sustainability of egg production. Both ingredients are byproducts of industrial productions, and the cultivation processes can sequester carbon from the atmosphere. Containing high levels of omega-3 polyunsaturated fatty acids (n-3 PUFAs), previous research reported successful egg n-3 PUFA enrichments when supplemented hempseed meal or microalgae independently. Such eggs can be marketed as specialty eggs to improve profitability of farmers and nutrition status of the public. Our preliminary research found promising results on reducing manure ammonia (NH3) production associated with the supplementations. However, with complementary n-3 PUFA compositions, potential synergistic effects of combined supplementation have not been investigated. Therefore, the objectives of the proposed work are to reveal the maximal potentials of feeding laying hens with an optimal combination of hempseed meal and microalgae on egg n-3 PUFA enrichments and manure NH3 reduction. Laying hens will be fed different supplementation combinations, and egg fatty acid profiles as well as manure NH3 levels will be assessed. We expect the combined supplementation of hempseed meal and microalgae will further improve egg n-3 PUFA enrichments than singular supplementation of either ingredient. Such findings will promote sustainability and resilience of the egg industry.
1. To investigate the enrichment levels of n-3 PUFAs and DHA in eggs through singular or combined supplementations of
hempseed meal and microalgae in laying hens.
Justification: Alongside the sustainable agriculture initiative, the added nutritional benefits of egg enrichments with n-3 PUFAs is another compelling aspect of supplementing hempseed meal in laying hens’ diets. However, previous research and our preliminary trial have reported that the most enriched n-3 PUFA is ALA with minimal enrichments of VLC n-3 PUFAs like EPA and DHA due to the fatty acid compositions of hempseed meals. On the contrary, supplementations of microalgal biomass with high concentrations of EPA and (or) DHA in laying hens can successfully biofortify such VLC n-3 PUFAs in the eggs accordingly. Therefore, the strategy of co-supplementation of hempseed meal and microalgae (Nannochloropsis oceanica) with complementary n-3 PUFA compositions is proposed to explore the optimal supplementation regime to maximize egg n-3 PUFA enrichments, which will provide added nutritional benefits to consumers and economic returns to poultry farmers.
2. To explore the impacts of hempseed meal and microalgae supplementations on the metabolism of laying hens and manure
NH3 production.
Justification: Due to their rich concentrations of fatty acids, antioxidants, and other bioactive compounds, supplementations of hempseed meal and microalgae may influence the metabolism of laying hens as well as modulate their gut microbial compositions.[37-39] Governed by microbiome, the enteric fermentation process in poultry produces short-chain fatty acids as well NH3, which the gas could be dangerous to animals and farmers when exposed to moisture and became ammonium.[40-42] Our previous research have shown encouraging results of reducing manure NH3 productions when supplemented hempseed meal and microalgae individually in broiler chickens and laying hens. Hence, the proposed work will examine the potential synergistic effects when both are supplemented concurrently in diets of laying hens for the health of animals and farm workers.
The purpose of this project is to explore the maximal potentials of feeding laying hens with an optimal combination of hempseed meal and microalgae as a sustainable regime to produce health-promoting eggs enriched with omega-3 polyunsaturated fatty acids (n-3 PUFAs) and to reduce manure ammonia (NH3) production.
Within the Northeast region, Pennsylvania is the 4th and New York is the 17th largest state of the U.S. for egg production totaling over 10 billion eggs produced in 2022.[1] Egg production is the third largest animal industry in Pennsylvania after dairy and beef cattle and the second largest in New York after dairy cattle.[2,3] With recent passage of the 2018 Farm Bill, there have been increasing interests in incorporating industrial hemp (Cannabis sativa L.) byproducts in animal agriculture like dairy cattle and poultry, for its promising sustainable agriculture initiative.[4,5] However, using hempseed as animal feed has not been officially approved by the U.S. Food and Drug Administration and state governments due to lack of scientific evidence for the safety of animals and food.[5,6] Therefore, further research is warranted to investigate the implications of feeding hempseed as a sustainable feed alternative. Furthermore, the value-added nutritional and environmental benefits provided by this practice necessitates further evaluation to empower egg farmers with the economic viability and holistic stewardship contributing to the Northeast SARE’s outcome statement.
Hempseed meal is a byproduct after the hemp heart processing and oil extraction.[7] It is a nutritious ingredient with high levels of protein, dietary essential amino acids, fatty acids, and antioxidants.[8-11] It can be a great substitute to the widely used soybean meal in the poultry industry, particularly when the U.S. production of soybean was predicted to decrease by 3.0% in 2036 compared with 2016 due to the changing climate.[12] Previous research has indicated that supplementing hempseed meal in broiler chicks and laying hens at relatively low inclusions (i.e., <20% of diets) did not affect animal health as well as meat or egg production.[13-17] Being an excellent source of n-3 PUFAs like alpha-linolenic acid (ALA), hempseed meal supplementations can modulate fatty acid compositions in animal products. Eggs produced from laying hens supplemented with hempseed meal were reported to contain noticeably higher concentrations of ALA as well as very-long-chain (VLC) n-3 PUFAs via endogenous fatty acid biosynthesis, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).[17-19] The use of microalgae from biofuel production in poultry diets has been comprehensively researched as a sustainable ingredient to replace corn and soybean meal. Influenced by the species, strains, and cultivating conditions, microalgae can be rich sources of VLC n-3 PUFAs; therefore, supplementations of such microalgae can enrich eggs with VLC n-3 PUFAs like EPA and DHA without negative effects on animal health, egg production, and egg characteristics.[20-23] However, studies investigating the potential synergistic benefits of combined supplementations of hempseed meal and microalgae have been lacking despite their complementary n-3 PUFA compositions.
Environmentally, hemp cultivations can absorb carbon dioxide (CO2) from the atmosphere and sequester carbon through biochar creation.[24,25] Agronomically, hemp is also a great candidate for crop rotation with a legume like soybean to improve soil fertility and suppress growth of weed, infectious agents, and nematodes.[25] Distinct from conventional feedstuff, microalgae are photosynthetic organisms inhabiting aquatic environment. The cultivation enables CO2 sequestration and mitigates the land-based food-feed competition, thereby rendering it an economically and environmentally sustainable process.[26-27] When supplemented in poultry diets, our previous research have suggested promising potentials of reducing NH3 emissions in broiler chicks and laying hens.
Repurposing hempseed meal and microalgae from byproducts to animal agriculture as valuable feed ingredients is a holistic practice that can reduce agricultural waste, decrease the food-feed competition, and promote sustainability and resilience of local small-scale egg farms. The proposed work will contribute to current research on hempseed and microalgae meaningfully by providing insights into animal health and egg enrichments of n-3 PUFAs, while also examining the potential climate benefits from reduced manure NH3 productions. Given the heightened awareness of the health benefits of n-3 PUFAs in the population, increased market values and demand for the enriched eggs may offset the extra cost associated with hempseed and microalgae supplementations. Therefore, it could improve the economic resilience and viability of egg farmers.
Research
Project objective 1: To investigate the enrichment levels of n-3 PUFAs and DHA in eggs through singular or combined supplementations of hempseed meal and microalgae in laying hens.
Study Design. A 6-week laying hen feeding trial with a 2-by-2 factorial design will be conducted at the Cornell University Poultry Research Farm (Ithaca, NY). In this proposed work, a total of 40 Shaver-White laying hens of peak egg production age will be recruited from Cornell University Poultry farm (Ithaca, NY) and randomly assigned into one of the four dietary treatment groups (n = 10 per group): 1) the corn-soybean meal basal diet as control, 2) the basal diet supplemented with 10% hempseed meal (IND HEMP, LLC., Fort Benton, MT), 3) the basal diet supplemented with 10% defatted microalgae Nannochloropsis oceanica (NO; Cellana, Kailua-Kona, HI), and 4) the basal diet supplemented with 10% hempseed meal and 10% NO. All diets will be formulated to be isocaloric and isonitrogenous meeting the nutrient requirements of laying hens per National Research Council’s guideline.[43] The supplementations of hempseed meal and microalgae in the treatment diets will largely replace the inclusion of soybean meal accordingly.
Animal husbandry and data collection. The laying hens will be housed in a temperature-controlled facility with free access to feed and water under the same husbandry conditions as previously described.[44] During the 6-week study, animal health status will be inspected daily either by the Research Farm staff or the graduate students. Board-certified poultry veterinarians from Cornell University’s College of Veterinary Medicine will also be available for consultations should it deemed necessary. To determine the impacts of hempseed meal and microalgae supplementations on egg production and characteristics, eggs will be recorded and collected daily, and egg components will be measured at the end of weeks 0, 3, and 6, including total egg weight, albumin weight, yolk weight, eggshell weight, and eggshell thickness. The body weight and feed intake of each hen will be recorded weekly. At weeks 0, 3, and 6, we will collect blood samples through brachial wing veins. Upon centrifugation, the plasma samples will be used to analyze health biomarkers, such as glucose, uric acid, and inorganic phosphorus. At the end of week 6, 5 representative birds within each treatment will be euthanized via CO2 asphyxiation per IACUC’s guideline for sample collections of liver (for fatty acid analysis) and cecal contents (for microbiome sequencing). Liver samples will also be archived in -80℃ freezer for additional analyses in the future should it be necessary, such as scenarios when plasma biomarkers suggesting potential hepatic damages or compromised metabolisms.
Fatty acid analysis. Freeze-dried egg yolk samples from baseline, week 3, and week 6 as well as liver samples will be extracted for lipid following the Folch method as previously described.[21,45] Briefly, approximately 0.25 g of liver or 0.10 g of dried egg yolk powder will be homogenized in Tris-EDTA buffer and followed by two subsequent extractions with 2:1 (v/v) chloroform-methanol and 4:1 (v/v) chloroform-methanol. Fatty acids will be methylated with methanolic sulfuric acid (1%). Methylated samples (i.e., fatty acid methyl esters; FAMEs) will be dried under nitrogen gas evaporator and re-suspended in hexane for quantification. The FAMEs will be quantified using a gas chromatography system (Agilent 6890N; Agilent Technologies) with a flame ionization detector and a fused-silica capillary column (CP-Sil 88, 100 m × 0.25 mm inner diameter, 0.2 µm film thickness; Agilent Technologies). The oven temperatures will be programed per Agilent’s recommendation.[44] Each FAME will be identified based on their respective retention times against the standard Supelco 37 Component FAME Mix (CRM47885; Sigma-Aldrich) and corrected using the internal standard tridecanoic acid (C13:0).
Project objective 2: To explore the impacts of hempseed meal and microalgae supplementations on the metabolism of laying hens and manure NH3 production.
Resting metabolic rate. To determine the impacts on animal metabolism, at weeks 3 and 6, we will randomly select 5 representative birds from each dietary treatment group for respiratory CO2 production analysis using our respiration chamber for poultry (0.25 m × 0.25 m × 0.30 m). Briefly, one bird at a time will be placed inside the chamber for 10-15 min, which a portable CO2 detector (CM-505; CO2Meter) will be connected to the chamber to record the CO2 production of the bird every 2 seconds. Total respiratory CO2 production (ppm) will be corrected with the animal’s metabolic body weight (g).
Fecal NH3 production and microbiome sequencing. At the end of weeks 3 and 6, fecal samples (approximately 100 g) from individual hens will be collected for NH3 measurements. Fecal samples will be stored in airtight plastic containers allowing microbial fermentations. Approximately 1.0 g of fecal samples will be subsampled for NH3 measurements immediately after original sampling, after 1 week of storage, and after 3 weeks of storage. Subsamples will be placed in an acrylic airtight box with a CO2 and NH3 multi gas detector (CM-507; CO2Meter) for 10 minutes. The detector measures CO2 and NH3 concurrently and records the data every 2 seconds. To reveal the fecal microbial compositions, fecal samples from the end of week 6 will also be used for 16S rRNA sequencing. Genomic DNA from the collected fecal samples will be extracted using Stool DNA extraction kit (QIAamp PowerFecal Pro DNA Kits; Qiagen) following manufacturer’s protocol and quantified with NanoDrop 1000 (Thermo Fisher Scientific Inc.). The 16S rRNA gene will be amplified using universal primers 8F and 515R with the primer sequences and PCR reaction conditions as previously described.[46] The PCR amplicons will be sequenced at a qualified facility such as the Genomics Core Facility of the Institute of Biotechnology at Cornell University. The obtained sequence reads will be processed and analyzed for alpha diversity, beta diversity as well as community composition as previously described.[46,47]
Statistical analysis: Data will be analyzed by two-way ANOVA to test for main effects of diets (i.e., hempseed meal, microalgae, and their interaction) and the Tukey’s HSD test for mean comparisons using the emmeans package in RStudio (R Foundation for Statistical Computing, Vienna, Austria). If any assumptions for two-way ANOVA were not met, non-parametric tests will be performed. Each cage (hen) will be the experimental unit. Statistical significances will be declared at P < 0.05.