Performing Department
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Non Technical Summary
Methane, a greenhouse gas that is 32 times more powerful in warming capacity than carbon dioxide, is attributed to 20% of global climate change, of which methane originating from the gastrointestinal tracts of domestic ruminants - such as beef and dairy cows - contributes nearly 10%. Enteric methane is not only burdensome to the planet, but is wasteful because it creates up to a 12% loss of energy for cattle (Johnson and Johnson, 1995). Despite the growing concern by the public, scientists, and industry, few practical solutions to this problem exist. There is an urgent need for additional research into methane suppression to produce and implement commercially viable solutions. While the planet is in desperate need for a solution that mitigates methane emissions, farmers and ranchers work on small margins and are extremely pragmatic about changes to their production systems, so introducing a technology that makes sense financially is essential.Feeding nitrate salts is an effective method to decrease enteric methane formation in ruminants. However, nitrate feeding, which is gaining traction in the cattle industry, is associated with a decrease in feed intake, which reduces production metrics of ruminants, undermining the sustainability of nitrate and disincentivizing its usage. Bezoar Laboratories LLC (Bezoar) aims to commercialize a new species of probiotic, Paenibacillus fortis, formerly 79R4, as its first product. When introduced to the ruminant's diet with nitrate, P. fortis quickly metabolizes the fed nitrate while enhancing it's methane eliminating potential (raising it from 47% to 54%). In addition, we project that the 10% increase in volatile fatty acids observed (VFAs) and reduction in methane that was observed in vivo will translate to around a 4% increase in overall feed efficiency and a 0.2% increase in product yield (Yan et al., 2010; Boyd et al., 2011). In dairy animals (n=18), nitrate plus Fortis increased milk yields by 5.7% above the control which was 1.7% higher than nitrate alone.Our formulation is in the prototype stage and has previously been tested in steers with a proof-of-concept trial in 2016 (Latham et al., 2019) and dairy cows in 2019-2020. However, additional research is needed to receive FDA approval of this novel feed additive. The research funded via the USDA SBIR Phase I program will be performed in conjunction with Texas A&M University and the USDA. It will include sixteen steers divided into four treatments ; control, nitrate alone, probiotic alone, and nitrate and probiotic together. The experiment will measure methane outputs of the animals. In addition, we will track the effect of P. fortis formulation on animal health and performance. Data resulting from the completion of these objectives will give the company firm numbers for the cost and benefits of implementing this technology and the overall benefits in reducing enteric methane formation. Preliminary estimates on revenue are around $85 per head per year. The cost to the producers will be approximately $22-$29 per cow/steer per year, offering our customers an outstanding ROI. This will effectively financially incentivize climate change remediation. For example, in Texas in 2012 there were 182 ranches with over 5000 cattle, with the mean number of cattle for this group being over 19,000 (USDA, 2012). If just ten of the average ranches utilized this novel feed additive, it would put annual revenue close to $5 million while taking the equivalent of 1 million cars off the road.
Animal Health Component
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Research Effort Categories
Basic
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Applied
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Developmental
100%
Goals / Objectives
Current knowledge leaves little doubt that nitrate can prevent enteric methane production in ruminants. The barrier to its widespread usage is associated with risks of nitrite poisoning and cost. Our invention, P. fortis, put forth by Bezoar, tackles many of the problems presented. Our formulation is in the prototype stage and has previously been tested in steers with a proof-of-concept trial in 2016 (Latham et al., 2019) and dairy cows in 2019-2020. Paenibacillus fortis is a denitrifying, spore-forming bacterium, isolated from the bovine rumen and selected for enhanced nitrite-reducing ability. Initial data shows that it enhances the methane mitigation potential of administered nitrate, reduced foodborne pathogens, increases fermentation efficiency, and enhances ruminal nitrite/nitrate detoxification. Therefore, it has the potential to reduce all adverse effects of nitrate supplementation in ruminants.However, Bezoar Laboratories requires a large in vivo experiment in beef steers to take this product to market. After completion of the experiment proposed in this USDA Phase I SBIR, Bezoar will submit all necessary documents to the FDA and AAFCO for approval as a feed additive. This process is estimated to take two to three years. Following the in vivo trial laid out in this SBIR Phase I, this lab group plans on implementing a production level pilot trial within 2021-2022 using Phase II SBIR funds. This will allow us to accurately calculate benefits in feed efficiency, morbidity and mortality, meat quality and quantity, microbiome changes and full necropsies. If results are successful, this will provide significant economic, social, and environmental benefits to the producers, end-users and the country as a whole.The experiment proposed has the following research objectives: 1. Validate methane-lowering ability of P. fortis formulation in a larger scale feeding study 2. Evaluate the effect of P. fortis formulation on animal health and performance including nitrate/nitrite poisoning and production efficiency respectively. Data resulting from the completion of these objectives will give the company practical numbers for the cost and benefits of implementing this technology. Farmers and ranchers work on small margins and are extremely pragmatic about deploying new technologies. Therefore, ensuring that the technology is economically viable will be key to actually getting the product to market.
Project Methods
The study consists of two 49 d periods. Each period will have a 28 d treatment application period and a 21 d adaptation followed by 7 d sample collection. The 7 d sample collection period is scheduled to include 5 d for measures of intake, digestion, and nitrogen balance, 1 d for gas exchange, and 1 d for ruminal fermentation parameters (rumen fluid samples will be collected at h 0, 2, 4, 8, 12, 16 and 24 after feeding). During the 21 d adaptation period rumen fluid will be collected at h 4 on days 1, 3, 7, 14, and 21. Blood will be drawn at h 4 on days -1, 1, 2, and 3 d to determine methemoglobin. Steers will be weighed regularly to assess average daily gain. Steers will remain on the growing diet for a 21-d withdraw period prior to the second period, to prevent carry-over effects. During the 21-d withdrawal period, no measures of intake, digestion, or ruminal fermentation will be conducted.On day 28, at h 0, 2, 4, 8, 12, 16 and 24 hours, rumen fluid will be collected with a suction-strainer (Raun and Burroughs, 1962; 19 mm diameter, 1.5 mm mesh) from three locations within the dorsal and ventral sacs of the rumen for analysis of pH, volatile fatty acids (VFA), in vitro ruminal CH4-producing rate, and ruminal ammonia-N, NO2 , and NO3. A portable pH meter (Symphony, VWR, Radnor, PA) will be used to measure pH of rumen fluid at the time of sampling. Subsequently, an 8 mL subsample of ruminal fluid will be combined with 2 mL of freshly prepared 25% (wt/vol) metaphosphoric acid, and then frozen at -20°C for later determination of VFA. An additional 9 mL subsample of ruminal fluid will be combined with 1 mL of 1 N HCl, then frozen at -20°C for later determination of RAN.A separate ruminal fluid sample will be collected prior to feeding the treatments on d 1 and at 4 h after feeding on d 1, 3, 7, 14, 21, and 28 to determine rate of ruminal NH3 production and CH4-producing rate. This sample is obtained by removing ruminal contents from three locations within the dorsal and ventral sacs of the rumen, strained through four layers of cheesecloth, and into insulated containers. The containers are filled with ruminal fluid and a small amount of ruminal contents then capped to reduce oxygen exposure. They are then immediately transported to the USDA microbiology laboratory. At the USDA, ruminal fluid and contents are blended using an IKA Lab Egg (Wilmington, NC) to dislodge microorganisms attached to the fibrous mat. Aliquots will then be apportioned for analyses of rate of ruminal NH3 production and CH4-producing rate.In vivo gas exchange will be determined on d 27 using portable respiration calorimeter (headboxes) using open-circuit calorimetry. Steers previously trained to the headboxes will have access to feed and water during the 24 h period they are inside the calorimeter. Methane and carbon dioxide production and oxygen consumption will be determined for each steer. Gas concentrations will be determined as described by Nienaber and Maddy (1985) and energy metabolism using the equations developed by Brouwer (1965). Additional analysis may be performed, if warranted, based on the production of methane.Diet, ort, and fecal samples are dried in a forced-air oven (96 h, 55°C) and allowed to air equilibrate to determine partial DM. Samples are ground (No. 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ) to pass through a 1-mm screen. Diet, ort, and fecal samples are dried at 105°C for DM determination, then combusted for 8 h at 450°C for OM determination. Crude protein will be determined by analyzing samples for N using Dumas combustion with urine and fecal samples being analyzed wet to prevent volatilization. Crude protein is calculated as N × 6.25. Analyses for NDF and ADF are performed using an ANKOM fiber analyzer (ANKOM Technology, Macedon, NY) with sodium sulfite omitted. Total tract digestion coefficients are calculated for OM and NDF using procedures as described by Cochran and Galyean (1994).Ruminal fluid samples, previously prepared for VFA determination, are thawed and centrifuged at 15,000 × g for 10 minutes and supernatant fluid is collected and stored at -20°C for VFA analysis. Volatile fatty acid concentrations are measured, after thawing, using a gas chromatograph as described by VanZant and Cochran (1994).Ruminal fluid samples, previously prepared for RAN determination, are thawed and centrifuged at 18,000 × g for 10 min. Ammonia, nitrite, and nitrate are measured colorimetrically as previously described (Latham et al., 2018)Rate of NH3 production is determined by incubation of 2 mL freshly collected rumen fluid with 6 mL anaerobic dilution solution (Bryant and Burkey, 1953) containing 2% (wt/vol) Bacto Trypticase Peptone (Becton, Dickinson and Company, Sparks, MD). Duplicate 18 × 150 mm crimp top culture tubes flushed with 100% CO2 are sealed using rubber stoppers with aluminum crimps and incubated at 39°C. Immediately prior to (h 0) and 6 h after incubation a 0.5 mL sample is removed and frozen for later analysis.For analysis, frozen samples are thawed and centrifuged at 18,000 × g for 10 min at 22°C. Ammonia is assayed colorimetrically according to Chaney and Marbach (1962) as described previously. Rates of NH3 production are calculated by dividing the difference between 6 and 0 h NH3 concentrations by 6 h incubation time.In vitro ruminal CH4-producing rate is determined by incubation of 5 mL freshly collected rumen fluid with 5 mL anaerobic dilution solution (Bryant and Burkey, 1953) containing 60 mM sodium formate and 0.2 g finely ground alfalfa as described by Anderson et al. (2006). Duplicate 18 × 150 mm Balch tubes flushed with 50% H2- 50% CO2 are sealed using rubber stoppers and aluminum crimps and incubated 3 h at 39°C. A 1.0 mL gas sample is removed from the headspace of each tube after 3 h of incubation and analyzed for CH4 producing rate by gas chromatography on a Gow Mac thermal conductivity series 580 gas chromatograph (Gow Mac Instrument, Bridgewater, NJ) equipped with a HaySep Q column (60°C, 25 mL/min of Argoncarrier gas).Intake, digestion, rate of ruminal NH3 production, and greenhouse gas production will be analyzed using the MIXED procedure of SAS 9.3 (SAS Inst. Inc., Cary, NC). Terms in the model will include P. fortis, nitrate, P. fortis × nitrate, block, and period, with animal as a random effect. Ruminal fermentation parameters will be analyzed using the MIXED procedure. Terms in the model included P. fortis, nitrate, hour, all their interactions, and period. Treatment means will be calculated using the LSMEANS option. The pdiff function will be used to separate treatment means.Energy and nitrogen balance will be calculated based on intake of gross energy and nitrogen, respectively, and outputs of these nutrients in milk, feces, and urine. The economic implications of differences in feeding efficiency will be calculated. The amount of DFM and nitrate consumed will be assigned a fair market value using financial projections so that the additional costs of the formulation can be considered. This will allow for a comprehensive assessment of the economic impact of incorporating a P. fortis and nitrate formulation into the feed of steers. Alongside calculations designed to assess commercial viability from an economic standpoint, careful analyses of the environmental impact of the probiotic formulation will also be performed. The environmental impacts of a reduction in inorganic nitrogen excretion as a result of P. fortis will also be considered.