Dairy cow manure is increasingly concentrated due to consolidation of farming operations, and is increasingly treated as a potential hazard to human and environmental health. In the Northeast there is particular concern about what to do with manure during the winter months when it cannot be spread safely or effectively for fear of runoff. Building a storage facility can be costly – anywhere from $100 to $1000 per cow depending on the design – and farmers who spend the money to build safe containment structures rarely save enough money on commercial fertilizers to benefit financially from being environmentally responsible.
To address these issues, our group has developed a method for raising Housefly larvae (Musca domestica) on dairy cow manure. Insect larvae are high in protein and fat, have rapid generation times, and can be cultivated using waste streams. So-called “upcycling” of organic waste into valuable ingredients is gaining traction worldwide, and insects are a core component of the strategy. After processing by larvae, dairy cow manure retains 75% of its wet weight (95% of dry weight), 80% of organic nitrogen (75% total nitrogen), and 94% of phosphorus. Moreover, processed manure has fewer antibiotic resistant bacteria. Thus, the production of insect larvae from manure results in a modest reduction in the volume of manure and increased biological safety by reducing antibiotic resistance. There is little tradeoff for farmers in terms of loss of raw nutrients (after larval processing the manure can still be used as fertilizer), and insect larvae can be processed into feed ingredients.
In this project I will follow previous work from our group to its next practical step – demonstrating the economic utility of insect larvae produced in the manner in the Northeast context. The goal of this study is to demonstrate a use for insects raised on dairy cow manure that 1) has potential to be profitable, 2) reduces manure volume, providing environmental and public health benefits, and 3) paves the way for growth of new agricultural industries in the Northeast.
I will investigate the utility of insect meal (IM) as a feed ingredient for cultivated rainbow trout (Oncorhynchus mykiss). The appeal of IM to fish farmers and hatchery managers is clear: production of high value farmed fish species like trout and salmon is currently limited by reliance on fishmeal (FM) and fish oil (FO) as feed ingredients. FM and FO are finite resources being exploited at close to or above sustainable limits, meaning that prices for FM and FO have increased rapidly. There is thus a strong push from both industry and environmental groups to develop alternatives to FM and FO.
Initial results using IM as a supplementary feed ingredient are promising, and one hypothesis for this is that insects represent a more natural diet for fish than commonly used ingredients (e.g. soybeans). The combination of high protein content, favorable amino acid profile, and evolutionary adaptation by freshwater fish makes IM an ideal candidate feed ingredient. Additionally, a recent study by Ido et al. showed that dietary Housefly IM can protect red sea bream (P. major) against bacterial infection. This result has not been repeated, but bears investigating: pro-immune effects would greatly increase the value of IM.
1) Raise housefly larvae on organic dairy manure. Our group’s previous publication on production of housefly larvae (M. domestica) used manure from the Cornell teaching barn. To demonstrate this technique using manure from an actual dairy farm, we will partner with Jerry Dell Farm, a local organic dairy producer.
2) Produce diets for rainbow trout using insect meal (IM). Diets with both high and low inclusion of IM will be formulated to meet the nutritional requirements of juvenile rainbow trout (O. mykiss). Modern fish feeds typically contain some fishmeal (FM), with most protein coming from plant ingredients to save on cost. In this preliminary stage, we aim to demonstrate that IM is a superior replacement for fishmeal than the modern alternatives (soy protein concentrate and corn gluten meal). This design gives our experiment the best balance of relevance (using IM in the context of a realistic modern diet) and control (direct comparison of FM replacements). If these results are encouraging, future studies will investigate the feasibility of using IM to cut down FM content even further.
3) Conduct trout feeding trial using IM diets. Juvenile rainbow trout will be fed diets with both high and low inclusion of IM to evaluate nutritional value of these feeds. A secondary hypothesis we are interested in evaluating is that IM diets are less pro-inflammatory than diets with high levels of plant ingredients, owing to lower levels of pro-inflammatory omega-6 fatty acids and trout’s evolutionary adaptation to eating insects. In addition to growth parameters, specific assays of immune health and inflammatory stress will be conducted.
Immune challenge with Bacterial Coldwater Disease (BCWD). At the end of the feeding trial, juvenile trout will be exposed to a bacterial infection. The objective is to broadly screen for possible immune-stimulatory effects of insect meal, as these have been reported in recent work . BCWD is a common infection in salmonids in both the hatchery and aquaculture context. Mortality can be as high as 85% in fry and fingerlings  and fish that survive the infection often present with scoliosis and lordosis as a result of muscle fiber destruction. There is no commercial vaccine for this disease, therefore development of an IM diet that enhances resistance to BCWD would provide additional value for both hatcheries and commercial fish farms. Absence of immune effects would, however, still be an additional and important verification of the viability of IM ingredients.
Hypothesis: Insects raised on dairy manure are a nutritionally viable feed ingredient for farmed rainbow trout (O. mykiss).
Specific Aim: Determine whether inclusion of Housefly larvae raised from organic dairy cow manure in feed affects growth and immune response of juvenile rainbow trout (O. mykiss) to Bacterial Cold Water Disease.
1) Raising housefly larvae on organic dairy manure
Larva meal was produced as described in our group’s recent publication. Housefly pupae purchased from a distributor (Spider Pharm, Yarnell, AZ) were used to establish fly breeding colonies. Adult flies were kept in mesh cages and provided a mixture of dry milk and sugar (1:1) as a source of food. Drinking water was provided in a closed water container with a protruding cotton wick. Flies were kept in a room at 25 ± 2°C with a photoperiod of 12 hours per day.
Fresh manure from a local organic dairy farm was used as a source of substrate for oviposition. Every three to four days, a cup of fresh manure was placed in a fly cage and eggs collected 18-24 hours later. Eggs were diluted to a density of approximately 4-8 eggs per gram of manure and maintained in a high humidity chamber. After a few days, larvae migrated out of the manure container and fell into an underlying plateau. Larvae were collected daily and stored at -20°C.
2) Production of diets for rainbow trout using insect meal
Three fish diets were prepared: a standard diet, a 5% larva meal diet, and a 30% larva meal diet. The diets were formulated as described in Table 1. The required protein content for fingerling diet is 38% of dry weight, and our control diet represents a modern diet containing proteins from fishmeal (10%), soy protein concentrate (20%), corn gluten meal (18%), and wheat gluten (5%). More explanation of rationale for this diet design can be found in the feeding trial section below. Diets were prepared by mixing the different ingredients with sufficient water to form a dough, spreading the dough into a thin layer on baking sheets and and drying to completion (no further reduction in weight after 4 hours of drying) in an oven at 50°C. The dried feed was then crushed by hand and using a food processor and the resulting crumble was size-sorted using stacked sieves. Final feed composition has been verified by Brookside Laboratories (New Bremen, Ohio). Dried feed was aliquoted and stored in the dark at -20°C for long-term storage.
3) Trout feeding trial using insect meal diets
Rainbow trout (O. mykiss) will be used for these experiments as it is an important hatchery and aquaculture species for the United States. Approximately 450 fingerlings of 1-2 g were purchased from Beaverkill Fish Hatchery (Livingston Manor, NY). Upon arrival, fish were acclimated for 4 weeks, then randomly sorted in groups of 10-12, batch weighed, and distributed to 10 gallon recirculating tanks at a stable temperature of 10-12°C. Fish have been fed 2 times a day to a maximum of 3% body weight per day. All experimental procedures have been approved by the Cornell Institutional Animal Care and Use Committee (IACUC).
b. Diet Groups
During acclimation, fish are being maintained on a commercial diet for fingerlings (Ziegler Bros, Gardners, PA). After the acclimation period, fish will be randomly assigned one of 5 experimental groups comprised of the 3 experimental diets. Table 2 defines the 5 experimental groups. Briefly, the idea behind using 5 treatments is to control for long-term vs. short-term immune-stimulatory properties of the insect diets. Group 1 is the control group and will be fed the control diet for 8 weeks prior to receiving the immune challenge. Groups 2 and 3 will be fed, respectively, a 5% and 30% larva meal diet for 8 weeks prior to challenge, whereas groups 4 and 5 will be fed the control diet for 6 weeks before being switched to the 5% and 30% larva meal diets for 2 weeks prior to challenge.
The reasons for selecting these categories are as follows. First, we wish to conduct an eight-week feeding trial comparing IM to plant ingredients at both high and low replacement levels (groups 1-3). Second, we wish to test the protective effects of these different diets against bacterial infection. Third, we wish to determine if the putative protective effects of larva meal are acquired rapidly (2 weeks), and whether they are sustained over longer feeding (8 weeks). Each group will have 84 fish spread across 6 technical replicates (6 tanks with 14 fish) per treatment. This design allowed us to conduct a well-controlled feeding trial that also served as a broad screen for immune-stimulatory effects of IM diets.
c. Data Collection
During the feeding trial, fish will be batch-weighed by tank every two weeks. Additionally, at the conclusion of the feeding trial blood, tissue, histological, and fecal samples will be collected from 10 fish per diet group and will be analyzed as follows:
Blood samples: lymphocyte counts will be performed; serum will be collected for antibody titer determination and quantification of pro-inflammatory cytokine levels.
Intestinal content: samples will be processed for bacterial DNA extraction for future analysis of the microbiota. Spleen tissue samples will be stored in Trizol and processed for detection of cytokine transcripts. Anterior kidney samples will be used to collect macrophages, and the phagocytic activity of the macrophages will be assayed using a standard bead assay.
Intestine, liver, and heart samples will be fixed in formalin for histological examination of inflammation.
This project is still in progress. Initial stages have gone smoothly and according to the timeline proposed in the application. Larvae production using the method established in Hussein et. al (2017) was successful, and the analysis of the diets has confirmed that they are consistent with the desired nutritional values of the experimental design. The fish are healthy and have acclimated well to our tanks and the dose determination trial is underway. More data will be presented in subsequent reports.
Education & Outreach Activities and Participation Summary
The progress of this ongoing project has been reported twice in combined lab meetings, as detailed in the grant application. Plans for subsequent outreach are on hold until conclusions of the work are known.
A manuscript will be submitted to a scientific journal to report results from this study.
Initial outreach efforts beyond the academic community will be directed toward the dairy, fish hatchery, and aquaculture industries in the following ways:
1) This project will be submitted for presentation at the Cornell Dairy Center of Excellence Symposium. These events are held at Cornell twice a year and emphasize communication between farmers and scientists.
2) This project will be submitted for presentation at the 2018 National SARE Conference. A column will be submitted to the aquaculture trade newspaper, Fish Farming News, to describe the benefits of this technology for hatcheries and fish farmers.
3) An article about this project intended for the general public will be submitted to New York State Conservationist magazine, a bi-monthly periodical put out by the Department of Environmental Conservation. In consultation with the editorial staff of the magazine and if allowed, this article will be submitted to similar state and national publications.