Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
NFREC
Non Technical Summary
This is submitted as a PREDOCTORAL application on behalf of Darren Henry (PD) by Dr. Nicolas DiLorenzo (PRIMARY MENTOR) from the Univeristy of Florida. This RESEARCH project addresses the Foundational Program Area: Animal health and production and animal products, and the Challenge Area: Agricultural and Natural Resources Science for Climate Variability and Change. The overall objective of this study is to mitigate CH4 production in beef cattle whilst maintaining a high level of productivity. The approach will consist of feeding a source of non-protein nitrogen (calcium-ammonium nitrate) as an alternative hydrogen sink to decrease enteric methane production in beef cattle. In vitro ruminal fermentation batch cultures will be conducted to evaluate the dosage effects of four inclusion rates of along with a negative control with no added non-protein nitrogen. To establish the impact of nitrate supplementation on ruminal metabolism and microbial populations, eight ruminally cannulated steers will be used in a 4×4 duplicated-Latin square. A third experiment will be conducted, where 24 growing heifers will be used to determine in vivo CH4 production and nutrient digestibility when supplemented with nitrate. Finally, an experiment evaluating the effects of nitrate on animal performance will be conducted with 192 growing cattle. Implications of this study may impact the global beef industry by providing information on a novel non-protein nitrogen source with the potential of increasing production efficiency, while decreasing the carbon footprint. This fellowship will be used to help prepare Darren to become successful in academia by allowing him to perform groundbreaking research.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Goals / Objectives
The ruminant animal is instrumental in producing the world's protein supply through meat and milk. It has been reported that of all the solar energy captured by the earth's biomass, only 5% is potentially available for human consumption (Russell and Gahr, 2000). The majority of the remaining 95% can be consumed by ruminants, fermented and broken down by ruminal microorganisms into products such as volatile fatty acids (VFA), which are then used as energy by the host ruminant to produce meat and milk (Russell and Gahr, 2000). One disadvantage of the fermentation of feeds to VFA is the production of reducing equivalents, which, in turn, need to be disposed of. Disposal of reducing equivalents is performed by transferring electrons and hydrogens to various acceptors in the rumen. Methane, which is formed by the reduction of CO2 or formate, is one of the most common and important hydrogen sinks in the rumen (Russell and Gahr, 2000). Methanogens, a subgroup of Archaea, use hydrogen as an energy source to form CH4, which reduces the concentration of hydrogen in the rumen to allow for rapid fermentation of substrates (Buddle et al., 2011). Regardless of the importance of CH4 as a hydrogen sink in the rumen, the production of CH4 can be detrimental to the environment by adding to the already elevated amounts of greenhouse gases (GHG). In general, GHG are measured in equivalents of CO2 (CO2eq) and CH4 is considered to be 20-25 times more potent as a GHG than CO2 (FAO, 2013). Enteric CH4 production in ruminants varies among diets of differing levels of concentrate, digestibility, and other aspects. At large, high-forage diets produce 3 to 4-fold more CH4 than high-concentrate diets (Johnson and Johnson, 1995). Nearly 70% of the energy requirements for cattle are used prior to weaning, when the principle dietary component is forage (Shike, 2013); therefore, reducing enteric CH4 production is a vital step in providing enough animal protein for the growing population while preserving our environment. Various life cycle assessments investigating several cattle production systems have reported that the largest influence on the carbon footprint of beef and dairy production is represented by on-farm emissions, when cattle are generally grazing (Beauchemin et al., 2010; Kristensen et al., 2011). When evaluating beef production specifically, approximately 80% of GHG emissions are released from the cow-calf phase of production (Beauchemin et al., 2011). It has been estimated that a greater than 6% increase in global enteric CH4 production will occur between 2015 and 2020 (2204 to 2344 MtCO2eq, respectively). This is a 32% increase from the year 1990 (EPA, 2006). Methane not only has an effect on the environment, but also on energy losses in cattle. It has been reported that CH4 accounts for up to 12% of gross energy losses in cattle (Johnson and Johnson, 1995). The percentage of energy lost is highly related to the type of diet provided. Losses from forage-based diets typically are 2-fold greater than energy losses to CH4 from cattle consuming high-concentrate diets (Harper et al., 1999; Doreau et al., 2011). If we consider that the 4.7 million beef cows in the southeastern US producing approximately 300 g/d of methane grazing forages, this accounts for 12.9 Tg of CO2eq each year. This is approximtely the same amount of GHG that the entire bus industry in the US produces. Studies focusing on cattle consuming diets with at least 50% concentrate have shown marked reductions in CH4 production of between 20 and 30%. Conservatively, assuming nitrate will reduce CH4 prodction by 15%, this will reduce GHG by nearly 2 Tg of CO2eq, which is equivilent to the amount of GHG produced by a small country such as Iceland. The goal of this project is to reduce the hazordous impacts of beef production on the environment. The data we collect will be beneficial and essential for not only researchers in the future, but also for policy makers when leading our country to be a self sustaining, environmentally healthy nation. Law should be based on facts and science, and this project will be one step in insuring that our population has a sustainable food supply.Objective #1: Evaluate the optimum inclusion rate of nitrate for methane mitigation using in vitro ruminal fermentation.Objective #2: Determine the effects nitrate on in vivo ruminal fermentation, metabolism, microbial ecosystem, and enteric CH4 production when feeding a low-quality forage diet. Objective #3: Assess the effect of nitrate on performance of growing cattle consuming a low-quality, forage-based diet.
Project Methods
Objective #1: Evaluate the optimum inclusion rate of nitrate for methane mitigation using in vitro ruminal fermentation.Experimental design, animals, substrates, and in vitro incubation: Objective 1 will be tested using a randomized complete block design with replicated in vitro fermentation analysis conducted on three separate days, with day considered as block. Treatments will include four inclusion rates of nitrate (0.0, 1.2, 2.4 and 3.6% of substrate DM), which will all be isonitrogenous using urea as a NPN source, and a negative control, which will not receive any added NPN. Two ruminally cannulated donor steers will be used to collect ruminal fluid for the in vitro incubation. Doses of nitrate are based on in vivo studies using cattle consuming high-concentrate diets (van Zijderveld et al., 2011; Newbold et al., 2014). Steers will be kept at the UF-IFAS North Florida Research and Education Center (NFREC) and will have ad libitum access to bahiagrass hay and water for at least two weeks prior to the first collection of ruminal fluid. Steers will be supplemented with 2.27 kg of molasses daily to mimick the in vitro substrate. A 4:1 McDougall's buffer:ruminal fluid mixture will be used as inoculum (McDougall, 1944). Substrates will be composed of bahiagrass hay and molasses. Treatments will be made isonitrogenous, using urea to replace calcium-ammonium nitrate, while providing similar degradable intake protein (DIP). Incubations will consist of 1.4 g of substrate (DM basis) with 100 mL of inoculum for 48 hours at 39°C under constant agitation (60 rpm) in 250-mL bottles, and gas production kinetics will be recorded using the Ankom Gas Monitoring System (Ankom Technologies, Macedon, NY). Three 48 hour periods (replicates) of in vitro incubations will be performed. After recording the final pH, 1 mL of 20% H2SO4 solution will be added to each bottle and subsequent 10 mL aliquots will be taken and stored at -20°C until analysis. Analyses will include CH4, N2O, H2S, and VFA production and NH3-N concentration of the inoculum. In vitro organic matter digestibility (IVOMD) will be performed and analyzed using a modified Tilley and Terry (1963) procedure.Objective #2: Determine the effects nitrate on in vivo ruminal fermentation, metabolism, microbial ecosystem, and enteric CH4 production when feeding a low-quality forage diet. Experiment 1:Experimental design, animals, diet and treatments: Eight ruminally cannulated Angus crossbred steers, from the NFREC herd in Marianna, FL, will be used in a duplicated Latin square design with four treatments to determine the effects of nitrate supplementation on ruminal fermentation and metabolism. Treatments include three inclusions of nitrate (determined from Objective 1) and a negative control (basal diet with no added NPN). Each of the four periods will be 22 days in length with day 0 to 13 for adaptation to treatments, day 14 for 24-h blood and ruminal fluid collection, day 16 to 17 for continuous pH measurements and day 18 to 22 for a washout period. From day 0 to 13, steers on the nitrate treatments will be adapted to the nitrate as described by Newbold et al. (2014): day 0 to 1 will provide 20.0% of total nitrates, day 2 to 3 will provide 40.0% of total nitrates, day 4 to 5 will provide 60.0% of total nitrates, day 6 to 7 will provide 80.0% of total nitrates, and day 8 to 13 will provide 100.0% of total nitrates.Steers will be housed in the NFREC Feed Efficiency Facility (FEF) and will have access to water and bahiagrass hay ad libitum and 2.27 kg/d of molasses. Molasses will also be used as a carrier for the treatments and urea. Steers not receiving nitrates will receive urea to provide a diet isonitrogenous to the diets containing nitrates. This diet is designed to provide a sufficient DIP to support ruminal microbial CP synthesis and growth (Galyean, 1996; Patterson et al., 2003), so the effect of nitrates as a NPN source can be tested against urea, a commonly used NPN source with no effects on methane emissions. Samples of water, hay and molasses will be tested for nitrate concentration prior to the onset of the experiment to determine total nitrate intake. Intake of hay will be recorded using the GrowSafe (GrowSafe Systems Ltd., Airdrie, Alberta, Canada) individual feed intake system, while liquid supplementation will be weighed and provided separately, daily, and orts will be recorded daily.Blood will be analyzed for blood urea nitrogen. Ruminal fluid and solids will be used to extract and analyze DNA of the microbial population using the Illumina MiSeq sequencing platform (Illumina, Inc., San Diego, CA). Furthermore, from the ruminal fluid, VFA and NH3-N concentration will be analyzed, along with pH.Experiment 2: Animals, diets and treatments: Twenty-four Angus crossbred heifers, from the NFREC herd will be used in a randomized complete block design with four treatments and two experimental periods (n = 12/treatment). Treatments will include the same three inclusions of nitrate from Objective 2 - Experiment 1 and one negative control (basal diet with no added of NPN). Each period will consist of a 14-day adaptation to treatments, five days of enteric CH4, and four days of feed and fecal collection to determine apparent total tract nutrient digestibility. Adaptation to nitrates will follow the same protocol described in Objective 2 - Experiment 1. Heifers will be housed in the NFREC - FEF and will have access to water and the same diet as Objective 2 - Exp. 1. The liquid supplement will also be used as a carrier for the treatments.In vivo CH4 emissions will be measured using the SF6 tracer technique (Johnson et al., 1994). Apparent total tract digestibility will be determined using indigestible NDF as an internal marker.Calculations: Daily CH4 emissions will be calculated using the ratio of SF6 to CH4, with the predetermined release rate of SF6 from the rumen. Total tract digestibility of DM, OM, CP, NDF, and ADF will be calculated as follows: 100 - 100 × [(marker concentration in hay/marker concentration in feces) × (nutrient concentration in feces/nutrient concentration in hay)]. Objective #3: Assess the effect of nitrate on performance of growing cattle consuming a low-quality, forage-based diet.Experimental design, animals, diet and treatments: To determine performance of growing cattle consuming a low-quality, forage-based diet, a randomized complete block design with four treatments will be used. Treatments will include the same inclusions of nitrate from Objective 2 and one negative control (basal diet with no added NPN). This experiment will be conducted at the NFREC using 48 pastures (1.32 ha pasture-1). Each pasture will contain four growing heifers. Cattle will be weighed, stratified by body weight (BW) and sex, and randomly assigned to one of the four treatments. The experimental period will consist of a 14-day adaptation to the pastures and treatments and a 70-day experimental period. Adaptation to nitrates will follow the same protocol described in Objective 2 - Experiment 1. Cattle will have ad libitum access to water and the same diet as Objective 2 - Exp. 1 and 2. The liquid supplement will also be used as a carrier for the treatments.Cattle will be weighed on two consecutive days at the beginning and end of the experimental period (days -1 and 0), to determine initial and final BW, average daily weight gain, and every two weeks until the end of the experimental period to assess weight gain progress. On days 0 and 70, ultrasonography will be performed to determine longissimus muscle (LM) area and fat thickness. Blood and liver samples will be collected to analyze mineral concentration of Cu, Zn, I, and Mn.