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The present thesis consists of two chapters. The first chapter focuses on reviewing previously conducted research pertaining to reducing direct, enteric methane (CH4) emissions from the California beef cattle industry to meet state regulatory goals and climate neutrality. The second chapter constitutes a study aimed to reduce enteric CH4 production through the use of a tannin containing feed additive. Forty-eight black Angus and black Angus x Herford heifers (body weight (BW) = 592 kg ± 11.0) were used in a randomized complete block design (n=24) and blocked based on individual, co-variate CH4 (111.0 g ± 7.2) emissions collected on day (d) -18 to -15. The two treatments that were used in this study were as follows: Control (CON; 48.87 g of ground corn/hd/d + 1.13 g Melengestrol Acetate (MGA; g/hd/d; Zoetis, Parsippany, NJ)) and Quebracho condensed tannin (TAN; 0.15% of dry matter (DM) + 48.87 g of ground corn/hd/d + 1.13 g MGA). Heifers were precision fed based on 2.2% BW and % DM of the total mixed ration (TMR) being fed. Individual dry matter intake (DMI) and water intake (WI) were monitored using the Roughage Intake Control System (RIC System; Hokofarm Group, Marknesse, The Netherlands). Enteric CH¬4, carbon dioxide (CO2), and hydrogen (H2) gas emissions were collected using the Green Feed (GF; C-Lock, Inc, Rapid City, SD). Enteric emissions were collected on d 10-13, 24-27, 38-41, 52-55, and on d 66-69. Sixteen, 3-hour sampling windows were staggered across a 90-hour sample period to collect a total of eight emission readings/animal/sampling period to determine the average 24-hour emission production from each individual heifer. Heifers were harvested on d 76, and individual carcass data was collected on d 77. Methane, CO2, and H2 production did not differ between TAN vs. CON heifers (P > 0.05). Methane and H2 yield were similar between TAN and CON fed heifers (P > 0.05). Carbon dioxide yield had a tendency (P = 0.075) to be reduced from heifers fed TAN vs. CON by 7.47% from d 38 to 41 (Table 15). Quebracho condensed tannin did not have an effect on enteric CH4, CO2, and H2 intensity in TAN fed heifers vs. CON fed heifers (P > 0.05). Body weight and average daily gain (ADG) were similar between heifers fed TAN vs. CON (P > 0.05). There were no differences between heifers fed TAN and heifers fed CON for gain to feed ratio (G:F), DMI, and WI (P > 0.05). Hot carcass weight (HCW), marbling score, preliminary yield grade (PYG), ribeye area (REA), and dressing percentage (DP) were similar between TAN and CON heifers (P > 0.05). All carcasses graded either Prime or Choice. In summary, precision supplementation of TAN at 0.15% of DM to feedlot heifers consuming a high concentrate did not affect enteric gas emissions, animal performance, and carcass characteristics and quality.

The objectives of the present thesis are to 1) review the current literature on enteric methane (CH4) mitigation interventions and 2) report the results and limitations of providing a feed additive to individual dairy cattle on a commercial farm. Chapter 1 considers the mode of action, efficacy, and moderating variables of anti-methanogenic interventions, as well as associated impacts on rumen fermentation, animal health and productivity, and net greenhouse (GHG) emissions. Selective breeding has the potential to produce lasting, cumulative CH4 emission reductions while dietary reformulation and feed additives generally offer immediate and reversible effects. Limited interventions are currently available for commercial use, and more on-farm research is needed before widespread implementation.Chapter 2 delves into an on-farm study, where the Agolin Ruminant (EOB) feed additive was pulse-dosed to dairy cows through GreenFeed (GF) units (C-Lock, Inc., Rapid City, SD). The primary parameters of interest included 1) enteric gases, particularly CH4 production (g/d) and CH4 intensity (g/kg milk or energy-corrected milk (ECM)), and 2) lactation performance. Seventy-two multiparous, mid-lactation cows were blocked by days in milk (DIM), parity, and milk yield and housed in a single pen. After a 2-wk covariate period, cows within each block were randomly assigned to one of two treatments for 10 weeks: Control (blank pellets) and EOBP (blank pellets + EOB pellets). Hopper 1 of each GF unit contained the EOB pellets, formulated to provide ~1g EOB from the first daily drop of pellets for each EOBP cow. Hopper 2 dispensed blank pellets to EOBP cows for their remaining daily drops and to Control cows for all drops. Enteric gases were measured via the GF units through open access and additional encouragement. Milk yields and components were obtained twice weekly, and individual dry matter intake (DMI) was modeled on a weekly basis using milk data, body weight, and body condition score. Overall, no significant differences were observed between treatments for any parameters, except for a tendency towards lower milk lactose concentration in cows receiving EOB. However, in week 7, the EOBP cows outperformed the Control cows in milk yield, ECM, yields of milk protein, lactose, and solids-non-fat, modeled DMI, and CH4 intensity (considering weekly CH4 production measurements from ≥ 2 time points). The lack of significant differences overall might be attributed to inherent limitations of administering EOB through the GF units, as well as a low average number of CH4 measurements (

California produces 2.55 billion pounds of almond kernels annually, and along with that 4.03 billion pounds of byproduct almond hulls are generated. Almond hulls have high sugar contents and are mainly used as livestock bedding and feed at present. The almond industry is seeking ways to reduce the environmental impact of conventional almond production by reducing dust generation and achieving zero waste. Off-ground harvesting is an emerging harvesting strategy that generates significantly less dust; however, off-ground harvesting produces a stream of wet hulls that can easily spoil if they are not processed properly. Wet almond hulls are an ideal feedstock for fermentation into high quality, probiotic-rich animal feed which can help the almond industry meet its zero-waste goal and increase revenue for almond farmers. Fermented animal feed is an alternative to the use of growth-promoting antibiotics in livestock and has already been recognized as an effective tool to improve gastrointestinal health and improve productivity in swine and poultry. It is possible that similar benefits can be observed in cattle fed fermented feed. Additionally, studies have shown that probiotic supplementation and alternative feeding strategies can reduce ruminant methane generation, the largest source of anthropogenic methane emissions in the US. The goal of this study is to assess the potential of using almond hulls as a feedstock for the production of a fermented cattle feed which can potentially reduce the environmental impact of the almond industry while concurrently increasing the value of almond hulls, improving cattle digestion, and reducing enteric methane emissions. In this study, California almond hulls of Nonpareil, Monterey, Independence, and Fritz varieties from a hulling facility were characterized for their chemical composition. The hulls had high sugar contents ranging from 31.8% to 42.2% by weight on a dry-basis (db) and phenolic compound content ranging from 3.4% to 7.6%, db. On- and off-ground harvested hulls of Independence, Monterey, and Fritz varieties were also characterized and compared. Off-ground harvested hulls had an average moisture content 3.5 times higher than on-ground harvested hulls of the same variety. Hulls were low in protein and fat at an average of 5.7% and 3.7% db, respectively and an average of 24.1% acid detergent fiber and 33.3% neutral detergent fiber. The hulls were characterized for the quantity of contaminants vs. hulls and on average, hull samples contained 88.9% hulls and 10.1% contaminants. Sieving and terminal velocity analysis were conducted to investigate methods for separating hulls from contaminants. The results showed that sieving separation was an effective method for separating hulls from contaminants. Solid-state, inoculated fermentation trials were conducted using Saccharomyces cerevisiae and Lactobacillus plantarum as inoculums. The effect of inoculum type, fermentation duration, inoculum amount, hull variety, particle size, and fermentation temperature were investigated through several fermentation experiments. It was found that fermentations using S. cerevisiae produced higher amounts of ethanol and acetic acid and caused an increase in pH, and fermentations using L. plantarum produced higher amounts of lactic acid and caused a drop in pH. Hull variety had a large impact on the characteristics of the fermented hulls under the conditions tested. 14-day fermentation durations produced similar results as 30-day fermentation durations in the conditions studied. Additionally, in-vitro digestion results indicated that almond hulls fermented with S. cerevisiae for 14 days reduced enteric CH4 production by 96% over 72 hours digestion at a 20% inclusion rate in a cattle diet.The nutritional composition of almond hulls, especially their high sugar content, makes them an ideal feedstock for the creation of fermented cattle feed. Almond hulls fermented with S. cerevisiae and L. Plantarum produce desirable characteristics for fermented feed including a pH below 4.5, high concentrations of lactic acid, and low concentrations of acetic acid. In-vitro digestion tests suggest that fermented feed is potentially an effective strategy to reduce enteric CH4 production.

Enteric fermentation represents the largest single source of anthropogenic methane (CH4) emission in the United States, accounting for 30% and 27% of the total CH4 emitted in California (CARB, 2020) and nationwide (US EPA, 2019), respectively. Due to the significant impact of CH4 on climate change and the negative correlation of animal productivity and enteric CH4 production, there is great interest in identifying feed additives that might mitigate CH4 synthesis in the rumen ecosystem. The first aim of this dissertation is to support enteric CH4 mitigation efforts by identifying novel feed additives that have significant potential to reduce enteric CH4 production from ruminant livestock. Red pigmented macroalgae, specifically members from the genus Asparagopsis, have previously been identified as feed additive that possess high potential of anti-methanogenic properties, however, has not yet been tested widely. The second aim of this dissertation was to test the efficacy of Asparagopsis taxiformis (A. taxiformis), a red macroalgae that has shown to reduce enteric CH4 when used in an Australian production environment, using an in vitro rumen simulation technique (RUSITEC) system to quantify the effects on enteric CH4 production and volatile fatty acid (VFA) production under feed regimes specific to California. When supplemented at a 5% organic matter (OM) dosage in vitro, A. taxiformis supplementation of CA specific cattle feed resulted in a 95% reduction in CH4 coupled with an increase in propionate to acetate ratio (Roque et al., 2019a). The third aim focused on the effects of the red macroalgae Asparagopsis armata (A. armata) on enteric CH4 synthesis and animal production parameters when fed to lactating dairy cows. A Latin square design was used with 3 treatment groups with 4 cows within each treatment and three 2-week treatment phases with 1-week washout periods between each treatment phase. Two doses of macroalgae were used (0.5% OM and 1.0% OM) in addition to the control group (0% OM) . Results from this study showed 26% and 67% decrease in CH4 production for 0.5% OM and 1.0% OM treatment groups, respectively. However, dry matter intake (DMI) was reduced by 10 kg/d between the control and 1.0% OM treatments, ultimately representing a reduction in milk yield of approximately 4 kg/d. Due to sorting behaviors observed in the dairy cows, we hypothesized that there was a taste aversion to high levels of macroalgae in the diet (Roque et al., 2019b). The fourth aim tested A. taxiformis macroalgae, similar to what was used in the in vitro study, fed to growing beef steers for 147 days. A randomized block design was used with 3 treatment groups (7 steers in each treatment). All steers were blocked by weight then randomly assigned to one of three treatment groups; control, 0.25% OM, and 0.5% OM. The objectives of this study were to determine the long-term (147 days) efficacy of macroalgae on reducing enteric CH4 and to determine if there were any effects on animal production. Results from this study indicate that A. taxiformis’ ability to reduce enteric CH4 persisted was more effective at reducing enteric CH4 compared to the (Roque et al., 2019b) dairy study and these effects persisted throughout the duration of the 147-day study. Additionally, reductions in DMI were also found in this study however, no changes in average daily weight gain (Roque et al., 2021). To further investigate macroalgae as a methane reducing feed additive, rumen fluid was collected from both in vivo studies and will be used to discover if there are changes happening to the rumen microbiome diversity and activity using metagenomic and metatranscriptomic approaches. The proposed objectives for future research are to observe the role that different rumen microbes play in methanogenesis as well as enteric fermentation in the rumen environment. Additionally, we will compare gene expression profiles of the rumen microbiome to determine which molecular processes are affected in the presence and absence of macroalgae.

Enteric fermentation represents the largest single source of anthropogenic methane (CH4) emission in the United States, accounting for 30% and 27% of the total CH4 emitted in California (CARB, 2020) and nationwide (US EPA, 2019), respectively. Due to the significant impact of CH4 on climate change and the negative correlation of animal productivity and enteric CH4 production, there is great interest in identifying feed additives that might mitigate CH4 synthesis in the rumen ecosystem. The first aim of this dissertation is to support enteric CH4 mitigation efforts by identifying novel feed additives that have significant potential to reduce enteric CH4 production from ruminant livestock. Red pigmented macroalgae, specifically members from the genus Asparagopsis, have previously been identified as feed additive that possess high potential of anti-methanogenic properties, however, has not yet been tested widely. The second aim of this dissertation was to test the efficacy of Asparagopsis taxiformis (A. taxiformis), a red macroalgae that has shown to reduce enteric CH4 when used in an Australian production environment, using an in vitro rumen simulation technique (RUSITEC) system to quantify the effects on enteric CH4 production and volatile fatty acid (VFA) production under feed regimes specific to California. When supplemented at a 5% organic matter (OM) dosage in vitro, A. taxiformis supplementation of CA specific cattle feed resulted in a 95% reduction in CH4 coupled with an increase in propionate to acetate ratio (Roque et al., 2019a). The third aim focused on the effects of the red macroalgae Asparagopsis armata (A. armata) on enteric CH4 synthesis and animal production parameters when fed to lactating dairy cows. A Latin square design was used with 3 treatment groups with 4 cows within each treatment and three 2-week treatment phases with 1-week washout periods between each treatment phase. Two doses of macroalgae were used (0.5% OM and 1.0% OM) in addition to the control group (0% OM) . Results from this study showed 26% and 67% decrease in CH4 production for 0.5% OM and 1.0% OM treatment groups, respectively. However, dry matter intake (DMI) was reduced by 10 kg/d between the control and 1.0% OM treatments, ultimately representing a reduction in milk yield of approximately 4 kg/d. Due to sorting behaviors observed in the dairy cows, we hypothesized that there was a taste aversion to high levels of macroalgae in the diet (Roque et al., 2019b). The fourth aim tested A. taxiformis macroalgae, similar to what was used in the in vitro study, fed to growing beef steers for 147 days. A randomized block design was used with 3 treatment groups (7 steers in each treatment). All steers were blocked by weight then randomly assigned to one of three treatment groups; control, 0.25% OM, and 0.5% OM. The objectives of this study were to determine the long-term (147 days) efficacy of macroalgae on reducing enteric CH4 and to determine if there were any effects on animal production. Results from this study indicate that A. taxiformis’ ability to reduce enteric CH4 persisted was more effective at reducing enteric CH4 compared to the (Roque et al., 2019b) dairy study and these effects persisted throughout the duration of the 147-day study. Additionally, reductions in DMI were also found in this study however, no changes in average daily weight gain (Roque et al., 2021). To further investigate macroalgae as a methane reducing feed additive, rumen fluid was collected from both in vivo studies and will be used to discover if there are changes happening to the rumen microbiome diversity and activity using metagenomic and metatranscriptomic approaches. The proposed objectives for future research are to observe the role that different rumen microbes play in methanogenesis as well as enteric fermentation in the rumen environment. Additionally, we will compare gene expression profiles of the rumen microbiome to determine which molecular processes are affected in the presence and absence of macroalgae.