Abstract
The red macroalgae (seaweed) Asparagopsis spp. has shown to reduce ruminant enteric methane (CH4) production up to 99% in vitro. The objective of this study was to determine the effect of Asparagopsis taxiformis on CH4 production (g/day per animal), CH4 yield (g CH4/kg dry matter intake (DMI)), average daily gain (ADG), feed conversion efficiency (FCE), and carcass and meat quality in growing beef steers. Twenty-one Angus-Hereford beef steers were randomly allocated to one of three treatment groups: 0% (Control), 0.25% (Low Dose; LD), and 0.5% (High Dose; HD) A. taxiformis inclusion based on organic matter intake. Steers were fed 3 diets: high, medium, and low forage total mixed ration (TMR) representing typical life-stage diets of growing beef steers. The LD and HD treatments over 147 days reduced enteric CH4 yield 45 and 68%, respectively; however, there was an interaction between TMR type and the magnitude of CH4 yield reduction. Supplementing the low forage TMR reduced CH4 yield 69.8% (P <0.001) for LD and 80% (P <0.001) for HD treatment. Hydrogen (H2) yield (g H2/DMI) increased significantly (P<0.001) 336 and 590% compared to Control for the LD and HD treatments, respectively. No differences were found in carbon dioxide (CO2) yield (g CO2/DMI), ADG, carcass quality, strip loin proximate analysis and shear force, or consumer taste preferences. DMI tended (P = 0.08) to decrease 8% in steers in LD treatment but significantly (P = 0.002) reduced 14% in steers in HD treatment. Conversely, FCE tended to increase 7% in steers in LD treatment (P = 0.06) and increased 14% in steers in HD (P < 0.01) treatment compared to Control. The persistent reduction of CH4 by A. taxiformis supplementation suggests that this is a viable feed additive to significantly decrease the carbon footprint of ruminant livestock and potentially increase production efficiency.
1 INTRODUCTION
Livestock production, particularly ruminants, contributes to anthropogenic greenhouse gas (GHG) emissions globally. These emissions are estimated to be 7.1 Gt carbon dioxide (CO2) equivalents annually, approximately 14.5% of the global anthropogenic GHG emissions (Gerber et al., 2013). The majority of GHG emissions from livestock production is mainly in the form of methane (CH4), which is produced largely through enteric fermentation and to a lesser extent manure decomposition. Enteric CH4 is a natural by-product of microbial fermentation of feed in the digestive tract especially in the rumen. Enteric CH4 emissions not only contribute to GHG but also represent an energy loss amounting up to 11% of dietary energy consumption (Moraes et al., 2014). Therefore, reducing enteric CH4 emissions contributes to the alleviation of climate change through reduction of GHG emissions from agriculture and can improve productivity through conservation of feed energy otherwise lost as CH4. There is potential for mitigation of enteric CH4 emissions through a variety of approaches with a focus on the use of feed additives, dietary manipulation and forage quality improvement (Hristov et al., 2013).
Feed additives used in CH4 mitigation can either modify the rumen environment or directly inhibit methanogenesis resulting in lower enteric CH4 production (g/day per animal) and yield (g/kg dry matter intake [DMI]). Reductions in CH4 production of beef cattle through the inhibition of methanogenesis have been reported for feed additives at 22, 93, and 98% for short-chain nitro-compounds (3-nitrooxypropanol; 3-NOP; Dijkstra et al. 2018), synthetic halogenated compounds (Tomkins et al. 2009), and naturally synthesized halogenated compounds in seaweed (Kinley et al. 2020), respectively. The compound 3-NOP inhibits the enzyme methyl-coenzyme M reductase which catalyzes the final step in methanogenesis in rumen archaea (Duin et al. 2016). Halogenated CH4 analogs, such as bromoform, act on the same methanogenesis pathway, but do so by binding and sequestering the prosthetic group required by methyl-coenzyme M reductase (Smith et al. 1962; Wood et al, 1968; Johnson et al., 1972). Some haloalkanes are structural analogs of CH4, and therefore competitively inhibit the methyl transfer reactions that are necessary in CH4 biosynthesis (Ermler et al., 1997; Liu et al., 2011). These CH4 analogues include bromochloromethane (BCM), bromoform, and chloroform and have been proven to be the most effective feed additives for reducing CH4 production. A 93% reduction of CH4 was shown in Brahman cattle with a feed inclusion of BCM at 0.30 g/100 kg LW twice daily for 28 days, however, there was no improvements on feed intake, weight gain, carcass quality or feed efficiency (Tomkins et al. 2009). Conversely, Abecia (2012) reported that the inclusion of BCM at 0.30 g/100 kg once per day decreased CH4 production 33% and increased milk production 36%. The authors speculated that increased milk production in BCM treated cows could be attributed to a shift to more propionate production in the rumen, which is a hydrogen (H2) sink and provides more energy compared to other volatile fatty acids. However, long-term efficacy of CH4 analogues in the rumen remains to be confirmed. Tomkins et al. (2009), for example, reported a second experiment resulting in a 57.6% CH4 reduction after 30 days of treatment which is far less than the reductions found during the first 28 days. Additionally, chloroform fed to fistulated dairy cows was effective at reducing enteric CH4 production through reduced abundance and activity of methanogenic archaea, but only over a 42 day period (Knight, 2011).
Types of feedstuffs can also drive CH4 production by providing different substrates to microbial populations which are the drivers of volatile fatty acid (VFA) production in the rumen. There are ways to influence the types of VFA produced in the rumen by changing the types of feed in the diet (Russell and Wallace, 1997, Van Soest, 1994). This is important for two reasons; first VFA represent the amount of energy available to the animal for means of animal productivity and second VFA pathways, such as the production of propionate, are able to utilize reducing equivalents that normally would be shifted to methanogenesis (Blaxter and Clappteron, 1965, Johnson and Johnson, 1995). Concentrates contain non-structural carbohydrates, such as starch and sugar, that are rapidly fermented which drives pH down, which negatively impact methanogenic populations, and are an effective way to increase propionate production (Bannink et al., 2006, 2008). Forages contain structural carbohydrates, such as neutral detergent fiber (NDF), and have been linked to CH4 production (Niu et al, 2018). As NDF in diet increases, rumen pH also increases resulting in preferential production of acetate over propionate, which generates reducing equivalents such as H2 that is shifted toward methanogenesis (Hungate, 1966, Janssen, 2010). Not only can NDF play a significant role in CH4 production, it has also been suggested to impact the efficacy of anti-methanogenic compounds added to feed (Dijkstra et al. 2018).
Red seaweeds, particularly the genus Asparagopsis, are considered potent anti-methanogenic organisms due to their capacity to synthesize and encapsulate halogenated CH4 analogues such as bromoform and dibromochloromethane within specialized gland cells as a natural defense mechanism against predation (Paul et al., 2006). Machado et al. (2014) compared a diversity of tropical macroalgae, including freshwater and marine species, and found that Asparagopsis taxiformis at 17% of OM had the largest reduction of CH4 production in vitro with a 98.9% average reduction. In the stepwise progression of evaluating the seaweeds Kinley et al. (2016a; 2016b) explored reduced inclusion rates to determine the in vitro minimum effective inclusion level of 2% of OM. In this process only A. taxiformis retained anti-methanogenic capability at a very low inclusion. A. taxiformis reduced CH4 production more effectively than synthetic halogenated CH4 analogs at equivalent concentrations in vitro, largely due to multiple anti-methanogenic bioactives working synergistically (Machado et al., 2018). Importantly, the concentration of bioactives in Asparagopsis spp, in particular bromoform, has a significant effect on the reduction of CH4 in animal trials. For example, Roque et al (2019b) tested the effects of the seaweed A. armata (1.3 mg bromoform/g DM) in vivo fed to dairy cattle for a two week duration and reported up to 67% reduction of enteric CH4 production using a 1% seaweed inclusion rate on organic matter (OM) basis in a total mixed ration (TMR). Kinley et al. (2020) reported up to 98% CH4 reduction using 0.2% of OM inclusion of A. taxiformis (6.6 mg bromoform/g DM) during a 90-day feeding regime typical of feedlot TMR. These studies confirm that seaweed quality measured as concentration of bioactive at feeding and the basal diet formulation have an impact on the efficacy of the seaweed, and that there is heightened response in vivo compared to in vitro.
For adoption of the seaweed by industry it is crucial that meat quality be maintained or improved. As with any feed additive, feeding A. taxiformis to livestock has the potential for changes in meat quality, tenderness and taste, and consumer acceptability. Marbling, for instance, directly impacts flavor and juiciness and it has been shown that marbling can directly influence consumer preference with some willing to pay a premium (Killinger et. al., 2004).
Therefore, the objectives of this study were to (1) measure the long-term effects of A. taxiformis over 147 day period, (2) test the efficacy of CH4 reductions of supplementing A. taxiformis to high, medium, and low forage basal TMRs, (3) quantify the effects of A. taxiformis supplementation on production parameters, meat quality (including taste), and bromoform residues within the meat and liver.
2 MATERIALS AND METHODS
This study was approved by the Institutional Animal Care and Use Committee of the University of California, Davis (Protocol No. 20803).
2.1 Study design, animals, and diets
Twenty-one Angus-Hereford cross beef steers, blocked by weight, were randomly allocated to one of three treatment groups: 0% (Control), 0.25% (Low Dose; LD), and 0.5% (High Dose; HD) inclusion rates of A. taxiformis based on OM intake. One steer on HD treatment was injured during the last 3 weeks of the trial and all data from this steer was removed from statistical analysis. The experiment followed a completely randomized design, with a 2-week covariate period before treatment began followed by 3-week data collection intervals for 21-weeks; a total of 147 days of seaweed treatment (Figure 1). During data collection intervals, alfalfa pellets offered through the CH4 measuring device (GreenFeed system, C-Lock, Inc., Rapid City, SD) were included as part of daily feed intake. The steers were individually housed and were approximately 8 months of age with an average BW of 352 ± 9 kg at induction to the trial. Steers were fed 3 diets during the study; high (starter diet), medium (transition diet), and low (finisher diet) forage TMRs, which are typical life-stage TMRs of growing beef steers (Table 1). Samples from the three diets and alfalfa pellets were collected once a week and bags of A. taxiformis were randomly sampled and analyzed (Table 2) for dry matter, acid detergent fiber, NDF, lignin, starch, crude fat, total digestible nutrient and mineral content (Cumberland Valley Analytical Services, Waynesboro, PA). Steers were offered water ad libitum.
Ingredients of the experimental diet containing high, medium, and low forage concentrations (% of DM)
Nutritional composition of experimental diets, Asparagopsis taxiformis, and alfalfa pellets
The A. taxiformis used as a feed additive was provided by Commonwealth Scientific and Industrial Research Organization (CSIRO) Australia. The seaweed was collected during the gametophyte phase from Humpy Island, Keppel Bay, QLD (23°13’01”S, 150°54’01”E) by Center for Macroalgal Resources and Biotechnology of James Cook University, Townsville, Queensland, Australia. Once collected, it was frozen and stored at −15 °C then freeze dried at Forager Food Co., Red Hills, Tasmania, Australia and later ground to a size of 2–3 mm. Total seaweed inclusion ranged from 46.7 to 55.7 g/day for LD and 76.1 to 99.4 g/day for HD treatment. The seaweed used in the study contained bromoform at a concentration of 7.8 mg/g dry weight as determined by Bigelow Analytical Services, East Boothbay, ME, USA. To increase palatability and adhesion to feed, 200 ml of molasses and 200 ml of water was added to A. taxiformis then hand mixed into the total mixed ration for each animal. The Control group also received 200 ml of both molasses and water with their daily feed to ensure A. taxiformis was the only difference between the three treatments. Steers were fed 105% of the previous day’s intake twice daily at 0600 and 1800 hours. Daily feed intake was calculated as the TMR offered minus feed refusal weights.
2.2 Sample collection and analysis
Methane, CO2, and H2 gas emissions from steers were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD, USA). Gas emissions were measured during the covariate period and experimental period during weeks 3, 6, 9, 12, 15, 18, and 21. In each measurement period, gas emission data were collected during 3 consecutive days as follows: starting at 07:00, 13:00, and 1900 hours (sampling day 1); 0100, 1000, and 1600 hours (sampling day 2); and 2200 and 0400 hours (sampling day 3). Breath gas samples were collected for 3 to 5 minutes followed by a 2-minute background gas sample collection. The GreenFeed system was calibrated before each measurement period with a standard gas mixture containing (mol %): 5000 ppm CO2, 500 ppm CH4, 10 ppm H2, 21% O2 and nitrogen as a balance (Air Liquide America Specialty Gases, Rancho Cucamonga, CA). Recovery rates for CO2 and CH4 observed in this study were within +/− 3% of the known quantities of gas released. Alfalfa pellets were offered at each sampling event as bait feed and was kept below 10% of the total DMI during each 3 day measurement period. The composition of alfalfa pellets is shown in Table 2. Feed intake and feed costs were recorded daily and bodyweight (BW) was measured weekly.
After the feeding trial was completed, all 20 steers were sent to a USDA-inspected commercial packing plant for slaughter. On the day of slaughter, steers were marked and followed throughout the process. On the first day, livers were collected and stored on dry ice until placed in a −20°C freezer. Carcasses were aged for 48 hours in a large cooler and then graded by a certified USDA grader. Directly after grading, carcasses were sent to fabrication where the strip loin from the left side of each carcass was cut and saved for further analysis. All 20 strip loins were placed on ice and transported back to the University of California, Davis where they were cryovac packaged and stored at 4°C in dark for 14 days. After 14-day of aging, strip loins were cut into steaks (2.45 cm thickness) and individually vacuum packaged and stored at −20°C. Steaks and livers were analyzed for bromoform concentrations using Shimadzu QP2010 Ultra GC/MS following a modified protocol described by Paul et al. (2006) (Bigelow Analytical Services, East Boothbay, ME, USA). The limits of bromoform detection and quantification were 0.06 mg/kg and 0.20 mg/kg, respectively.
To test for objective tenderness, slice shear force (SSF) and Warner-Brazler shear force (WBSF) were measured following the protocol described by AMSA (2016). One steak from each animal was thawed overnight and cooked to an internal temperature of 71°C. Within 1 to 2 minutes after cooking, the SSF were measured using machine texture analyzer (TMS Pro Texture Analyzer, Food Technology corporation, Sterling, VA, USA) with a crosshead at the speed of 500 mm/minute. To test WBSF, cooked steaks were cooled at 4°C overnight, and then four cores were cut using WEN 8-inch 5 Speed Drill Press from one steak from each animal parallel to the muscle fiber orientation. The WBSF was measured using the TMS Pro texture analyzer with a Warner Bratzler blade (2.8 mm wide) and a crosshead at speed of 250 mm/minute. The average peak forces for all four cores were recorded.
A consumer sensory panel was conducted at UC-Davis. Strip steaks were thawed at 4°C for 24 hours then cooked to an internal temperature of 71°C using a George Foreman clamshell (Spectrum Brands, Middleton, WS, USA). Internal temperature was taken from the geometric center of each steak using a K thermocouple thermometer (AccuTuff 351, model 35100, Cooper-Atkins Corporation, Middlefield, CT, USA). Following cooking, steaks were rested for 3 minutes then cut into 1.5 cm3 pieces. Each steak was randomly assigned a unique three digit number, placed into glass bowls covered in tin foil then stored in an insulated food warmer (Carlisle model PC300N03, Oklahoma, OK, USA) for longer than 30 minutes prior to the start of each sensory evaluation session. A total of 112 participants evaluated steak samples during one of the 5 sessions held over a 4-day period. Each participant evaluated a total of three steak samples, one from each treatment group, with a minimum of two 1.5 cm3 pieces per steak. Each participant was asked to evaluate tenderness, flavor, juiciness, and overall acceptance using a 9-point hedonic scale (1 = Dislike extremely and 9 = Like extremely).
2.3 Statistical analysis
Statistical analysis was performed using R statistical software (version 3.6.1; The R Foundation for Statistical Computing, Vienna, Austria). The linear mixed-effects models (lme) procedure was used with the steer as the experimental unit. GreenFeed emission data were averaged per steer and gas measurement period, which was then used in the statistical analysis. Dry matter intake and cost per kg of gain (CPG) data was averaged by week and used in the statistical analysis. Average daily gain (ADG) was calculated by subtracting initial BW from final BW then dividing by the number of experimental days for each diet regimen and the duration of the study (i.e. 63 days on high forage (starter) TMR, 21 days on medium forage (transition) TMR, then 63 days on low forage (finisher) TMR with total study duration of 147 days). Feed conversion efficiency (FCE) was calculated by dividing ADG by DMI for each diet regimen and the duration of the study.
The statistical model included treatment, diet, treatment × diet interactions, and the covariate term, with the error term assumed to be normally distributed with mean = 0 and constant variance. Individual animal was used as random effect, whereas all other factors were considered fixed. Data was analyzed as repeated measures with an autoregressive 1 correlation structure. Statistical significance was established when P ≤ 0.05 and a trend at 0.05 > P ≤ 0.10. The consumer sensory evaluation data were analyzed using the Kruskal-Wallis test. The Dunn’s test with P-value adjustment following Bonferroni methods was used for post-hoc pair-wise comparisons.
3 RESULTS
3.1 Gas parameters
The emissions as production (g/day per animal) and yield (g/DMI kg) of CH4, H2, and CO2 gases from the steers in the three treatment groups (Control, LD, HD) are presented in Figure 2 (for the duration of the trial) and Table 3 (divided by the three diet regimes). Where P values for significant effects are not given, they are P < 0.01. Inclusion of A. taxiformis in the TMR had a significant linear reduction in enteric CH4 production and yield. Methane production for the collective feeding stages of experimental period resulted in a reduction of 50.6 and 74.9% for LD and HD treatments, respectively, compared to Control. Methane yields for LD and HD were 45 and 68% lower, respectively, compared to Control. However, there was an interaction between diet formulation and magnitude of CH4 production and yield reduction. Methane production in steers on the high forage TMR with A. taxiformis inclusion was significantly reduced 36.4% for LD and 58.7% for HD and CH4 yield was significantly reduced 32.7% for LD and 51.9% for HD compared to the Control steers. Methane production during the medium forage TMR phase was significantly reduced 51.8 and 86.8%, for LD and HD treatments compared to Control, respectively, whereas CH4 yield was significantly reduced 44.6 and 79.7%, respectively. Steers fed low forage TMR in LD and HD treatments produced 72.4 and 81.9% lower CH4 compared to Control, respectively. Similarly, their CH4 yield was significantly reduced 69.8 for LD and 80.0% for HD.
Effect of Asparagopsis taxiformis inclusion levels of 0%, 0.25%, and 0.5% of feed organic matter to three stages of beef cattle diets on gas parameters
Hydrogen production significantly increased 318 and 497% and H2 yield also increased significantly 336 and 590% compared to Control for the LD and HD treatments for the collective feeding stages of the experiment, respectively. Hydrogen production in steers receiving A. taxiformis to high forage TMR in the LD and HD treatments significantly increased 177 (P = 0.03) and 360%, respectively. Hydrogen yield increased 198 (P = 0.04) and 478% in steers on LD and HD treatments, respectively. Hydrogen production from steers on the medium forage TMR increased 326 (P = 0.03) and 535% (P < 0.01), for LD and HD treatments, respectively, whereas H2 yield significantly increased 404 and 753%, respectively. Supplementation of A. taxiformis to the low forage TMR fed to steers significantly increased H2 production 419 and 618% and reduced H2 yield 503 and 649% in LD and HD treatments, respectively. Carbon dioxide (CO2) production was not affected by either LD or HD treatments compared to Control. However, CO2 yield was significantly greater in HD group compared to Control (P = 0.03).
3.2 Animal production parameters
Dry matter intake, ADG, feed conversion efficiency (ADG/DMI; FCE) and cost per gain ($USD/kg weight gain; CPG) as impacted by treatment groups (Control, LD, HD) for the collective feeding stages is presented in Table 4 and for the individual stages and TMRs in Table 5. Initial BW, final BW, carcass weight and total weight gained are shown in Table 4. Considering all feeding stages, steers on the LD treatment tended (P = 0.08) to decrease their DMI 8% and was significantly reduced 14% in steers on the HD (P < 0.01) treatment compared to Control. Steers fed the high and medium forage TMR in the HD treatment decreased their DMI 18.5 (P = 0.01) and 18.0% (P < 0.01), respectively, compared to Control. No significant effects were observed in ADG by the LD or HD treatment groups. With the reduction of DMI in LD and HD treatments and similar ADG among all 3 treatments, FCE tended to increase 7% in LD (P = 0.06) treatment and significantly increased 14% in steers in HD (P < 0.01) treatment. When averaged throughout the experiment as well as within the 3 TMR stages, CPG was not statistically significant. However, over the collective feeding stages, CPG was consistently lower in HD and LD groups compared to Control with approximately $0.37 USD/kg gain differential between HD and Control and $0.18 USD/kg gain differential between LD and Control. Additionally, cost differentials for HD were $0.29, $0.40, and $0.34 USD/kg gain and for LD were $0.15, $0.49, and $0.34 USD/kg gain for the high, medium, and low forage TMRs, respectively.
Effect of Asparagopsis taxiformis inclusion levels of 0%, 0.25%, and 0.5% of feed organic matter on animal parameters over 21 weeks
Effect of Asparagopsis taxiformis inclusion levels of 0%, 0.25%, and 0.5% feed organic matter to beef cattle diets on animal parameters
3.3 Carcass quality parameters
There was no statistical difference between treatment groups for rib eye area (Table 6). No effects were found between Control, LD, and HD in moisture, protein, fat, ash, carbohydrates, or calorie content of strip loins (Table 6). The average WBSF values for the Control, LD and HD groups were 2.81, 2.66 and 2.61 kg, respectively. Additionally, the SSF averages were measured as 17.1 for Control, 16.75 for LD and 17.4 kg for HD treatments. No significant differences (P > 0.05) were found in shear force resistance among treatment groups. Mean scores of all sensory attributes (tenderness, juiciness, and flavor) by consumer panels were not significantly different (P > 0.05) among treatment groups (Table 6). The taste panel considered all steaks, regardless of treatment group, to be moderately tender as well as moderately juicy. This was consistent with the taste panel stating that they moderately liked the flavor of all steaks regardless of treatment group. There was no difference (P > 0.05) in overall acceptability among treatment groups.
Effects of Asparagopsis taxiformis supplementation on carcass quality, proximate analysis, shear force, and consumer panel preference.
There was a linear increase in iodine concentrations in both LD (P < 0.01) and HD (P < 0.01) compared to Control. Iodine concentrations for the Control treatment group were below detection levels, which was set at 0.10 mg/g (Table 6). However, 5 out of 7 steers in LD treatment group had iodine levels above the detection level with a treatment average of 0.08 mg/g (P < 0.01). All 6 steers in the HD treatment group were found to contain iodine levels above the detection level with concentration levels ranging between 0.14 – 0.17 mg/g with a mean of 0.15 mg/g (P < 0.01). Bromoform concentrations for all treatment groups were below detection levels, which were 0.06 mg/kg.
4 DISCUSSION
4.1 Enteric methane production and yield
This study demonstrated that dietary inclusion of A. taxiformis induces a consistent and considerable reduction in enteric CH4 production from steers on a typical feedlot style diet. Enteric CH4 is the largest contributor to GHG emissions from livestock production systems. Similar reductions in CH4 yield, which is standardized by DMI, has also been established. There is a concern that feed additives and other CH4 reducing agents decrease in efficacy over time (Knight et al., 2011, Rumpler et al., 1986). This study provided evidence that the seaweed inclusion was effective in reducing CH4 emissions, which persisted for the duration of the study of 147 days (Figure 3). Notably, until this study the longest exposure to A. taxiformis had been demonstrated for steers in a study ending after a 90-d finishing period (Kinley et al. 2020). To date, only three in vivo studies have been published using Asparagopsis spp to reduce enteric CH4 emissions in sheep (Li et al. 2018), lactating dairy cattle (Roque et al., 2019b), and feedlot Brangus steers (Kinley et al., 2020). All studies show considerable if not variable reduction in enteric CH4 emissions. The differences in efficacy are likely due to levels of seaweed inclusion, formulation of the diets, and differences in seaweed quality based on bromoform concentrations.
It has been previously hypothesized that NDF levels can also influence the rate at which CH4 is reduced with the inclusion of inhibitors (Dijkstra et al. 2018). In the current study, the magnitude of reductions in CH4 production were negatively correlated (r2 = 0.89) with NDF levels in the 3 diet regimens that contained 33.1% (high forage), 25.8% (medium forage), and 18.6% (low forage) NDF levels. Enteric CH4 production was reduced 32.7, 44.6 and 69.8% in steers on the LD treatment and 51.9, 79.7, and 80.0% on HD treatment with high, medium and low forage TMRs, respectively. The low forage TMR, containing the lowest NDF levels, was the most sensitive to the inclusion of A. taxiformis with CH4 reductions above 70% at equivalent inclusion levels compared to the higher forage TMRs. Li et al. (2018) reported an 80.6% reduction of CH4 yield in sheep fed diets containing 55.6% NDF, however, the level of A. taxiformis intake by the sheep was unclear but was offered at 6 times greater levels than the HD treatment in our study. Roque et al. (2019b) showed 42.7% reduction in CH4 yield in lactating dairy cattle fed a diet containing 30.1% NDF at 1% inclusion rate of A. armata. The high forage TMR in our study had a similar NDF level to Roque et al. (2019b), however, had approximately double the reduction of CH4, even when consuming 50% less seaweed. These differences relate to a large degree to the quality of seaweed in terms of the concentration of bromoform, which was 1.32 mg/g in Roque et al. (2019b) compared to 7.82 mg/g in the current study. Kinley et al. (2020) conducted an in vivo study focused on feedlot steers using the same collection of A. taxiformis as sub-sampled and used in this study. This seaweed had bromoform concentration of 6.55 mg/g, which was marginally lower than our study and may be due to variation in the collection, sampling or analysis techniques. Despite the lower bromoform concentration in the seaweed and using 0.20% inclusion rate of A. taxiformis on OM basis, the CH4 yield was reduced by up to 98% in Brangus feedlot steers. The diet used by Kinley et al. (2020) included 30.6% NDF, which was similar to our high fiber diet.
The greater efficacy of A. taxiformis in that study could be due to collective feed formulation differences such as the energy dense component of barley versus corn, which is typical of Australian and American feedlots, respectively. Additionally, it could be due to beneficial interaction with the ionophore, monensin, that was used in the Australian study. Monensin has not been used in any other feed formulation in other in vivo studies with the inclusion of Asparagopsis species. Use of monensin in diets has shown to decrease CH4 yields by up to 6% in feedlot steers while also having an enhanced effect in diets containing greater NDF levels (Appuhamy et al., 2013). A potential enhancing interaction of the seaweed with monensin is of great interest and further investigation will elucidate this potential that could have significant beneficial economic and environmental impact for formulated feeding systems that use monensin.
4.2 Enteric hydrogen and carbon dioxide emissions
Increases in H2 yield have typically been recorded when anti-methanogenic feed additives are used, and with the addition of Asparagopsis species in dairy cattle (1.25 to 3.75 fold; Roque et al., 2019b) and Brangus feedlot steers (3.8-17.0 fold; Kinley et al., 2020). Similar increases in H2 yield have been reported in feed additives that reduce enteric CH4 emissions targeting methanogens. For example, in lactating dairy cows supplemented with 3-NOP, H2 yield increased 23 – 71 fold (Hristov et al., 2015). Bromochloromethane (BCM) fed to goats increased H2 (mmol/head per day) 5 – 35 fold, while chloroform fed to Brahman steers increased H2 yield 316 fold (Mitsumori et al., 2012; Martinez-Fernandez et al., 2016). Although feeding Asparagopsis spp. increased overall H2 yield (Figure 4), the magnitude was considerably lower (1.25 to 17 fold) compared to alternative CH4 reducing feed additives (5 to 316 fold), with similar levels of reductions in CH4. This indicates that there may be a redirection of H2 molecules that would otherwise be utilized through the formation of CH4 and redirected into different pathways that could be beneficial to the animal. For example, increased propionate to acetate concentrations have been recorded in in vitro studies using A. taxiformis (Machado et al., 2016a; Roque et al., 2019a) during inhibition of CH4 which could indicate that some of the excess H2 is being utilized for propionate production. Consistent with this theory, similar results have been reported using other CH4 analogues such as BCM (Denmen et al., 2015) and chloroform, (Martinez-Fernandez et al., 2016) both of which showed increases in propionate production. In general, significant reduction of CH4 production in the rumen without detriment to rumen function is typically associated with reduction of acetate, increase in propionate, and reduction of the acetate:propionate ratio (Kinley et al., 2020).
In contrast to the lactating dairy cattle study in which CO2 yield increased significantly in dairy cattle fed 1% A. armata (Roque et al., 2019b), there was no significant difference in CO2 emissions or yield in the current study. This could be due to the relationship between the amount of seaweed fed and DMI intake.
4.3 Animal production parameters
Dry matter intake reductions observed in this study were consistent with previous studies in lactating dairy cows where decreases in DMI were found to be 10.7 and 37.9% at 0.50 and 1.0% inclusion rate of A. armata (Roque et al., 2019b), respectively. Decreases in DMI have also been reported in cattle fed other anti-methanogenic feed additives in a linear dose-response fashion. For example, Tomkins et al. (2009) reported 3 to 19% reductions in DMI in steers supplemented with BCM at dosages between 0.15 and 0.60 g/100 kg live weight. Additionally, Martinez-Fernandez (2016) found 1.7 to 15% reductions in DMI when feeding steers chloroform at dosages between 1 to 2.6 g/100g liveweight. In contrast, Kinley et al. (2020) reported no significant differences at the highest A. taxiformis level of 0.20%. However, the inclusion level was less than our study’s lowest inclusion rate, so based on previous experiment’s observation of reduced DMI in a dose-response manner (Roque et al., 2019b), it was expected to have lower effect on DMI. Decreases in DMI are normally associated with lower productivity due to lower levels of nutrients and dietary energy consumed. However, there was no significant difference in ADG between steers in the HD treatment and Control (average 1.56 kg/day) groups despite consuming 14% less feed. The results were in agreement with a previous study (Roque et al., 2019b), in which milk production was not compromised at a 0.5% OM inclusion level despite reductions in DMI. The FCE (ADG/DMI) increased significantly in HD treatment group, suggesting that inclusion rates of A. taxiformis at 0.5% improves overall feed efficiency in growing beef steers. Since a large proportion of on farm costs is the purchase of feed, an improved feed efficiency is particularly exciting for producers to decrease feed costs while also producing the same amount of total weight gains. Total gains were between 224 kg (LD) to 236 kg (HD) combined with an average cost differential of ~$0.18 USD/kg gain (LD) and ~$0.37 USD/kg gain (HD). A producer finishing 1000 head of beef cattle has the potential to reduce feed costs by $40,320 (LD) to $87,320 (HD) depending on seaweed dosage. While the CPG in this study were not statistically significant, this may be due to low animal numbers in each treatment and warrants further investigation on a larger feedlot setting to reduce animal variability.
4.4 Bromoform and iodine residues
Bromoform is considered to be the active ingredient responsible for CH4 reduction when fed to cattle (Machado et al., 2016b). However, high levels of bromoform is considered to be hazardous for humans and mice. While bromoform intake limits are yet to be defined for cattle specifically, the United States Environmental Protection Agency (2017) has suggested a reference dose for bromoform, an estimated level of daily oral exposure without negative effects, to be 0.02 mg/kg BW/day. It is essential that food products from livestock consuming the seaweed are confirmed as safe for consumption and that bromoform residues are not transferred to the edible tissues and offal of bovines at levels detrimental to food safety. Previous studies have demonstrated that bromoform was not detectable in the kidney, muscle, fat deposits, blood, feces, and milk in either sheep (Li et al., 2018), dairy cows (Roque et al., 2019b), or feedlot steers (Kinley et al., 2020). Strip loin and liver samples from steers were collected and in agreement with previous studies, no bromoform was detected in this study.
The National Academies of Sciences, Engineering, and Medicine (2016) recommendations for daily iodine intake in growing beef cattle is 0.5 ug/g DMI and maximum tolerable limit is 50 ug/g DMI. In this study, recommended daily iodine intake levels were 5.2 mg/day and 4.85 mg/day and maximum limits are 521 mg/day and 485 mg/day for LD and HD treatment groups, respectively. The iodine level in the A. taxiformis fed in the current study contained 2.27 mg/g, therefore, maximum daily intake of seaweed iodine was 106 – 127 mg/day and 173 – 225 mg/day for the LD and HD treatment groups, respectively. While these levels do not exceed maximum tolerable limits, they exceed daily iodine intake recommendations, therefore it was appropriate to test for iodine residue levels in meat used for human consumption. The US Food and Nutrition Board of the National Academy of Sciences has set a tolerable upper intake level (UL) for human consumption of foods, which is defined as the highest level of daily intake that poses no adverse health effects (Trumbo et al., 2001). The iodine UL ranges between 200 ug/day to 1,100 ug/day depending on age, gender, and lactation demographics. Strip loins tested for iodine residues had levels of 0.08 and 0.15 ug/g from steers in treatments LD and HD, respectively. These iodine residues are far under the UL limits for human consumption. For example, UL for a person under 3 years of age is 200 ug/day meaning that this person would have to consume more than 2,500 g/day and 1,330 g/day of meat from a LD and HD steers, respectively, to reach the UL. An adult over the age of 18 has an UL of 1,100 ug/day and would have to consume more than 13.8 kg/day and 7.3 kg/day of meat from a LD and HD steers, respectively, to reach their UL of iodine intake. At the inclusion levels and iodine concentration of A. taxiformis used in this study the margin of safety is extremely high and the likelihood of iodine toxicity from consuming the meat is extremely low.
4.5 Carcass quality parameters
Marbling scores ranged from 410 – 810 while all carcasses, regardless of treatment, graded as either choice or prime. The value placed on tenderness in the marketplace is high and has even been found that consumers are likely to pay premiums for more tender beef (Miller et. al, 2001). Rodriquez-Herrera et al (2016) found that cattle supplemented with a high amount of Schizochytrium spp. (30 g/kg of feed intake) had improved tenderness compared to the control treatment and a low dose treatment group. Conversely, Phelps et al (2016) reported that supplementation of Schizochytrium spp. in cattle at 0 to 150 g/day per animal, a lower inclusion rate than Rodriquez-Herrera et al (2016), did not have a significant impact on objective tenderness. While no significant differences in meat tenderness were observed for either WBSF or SSF values among the three treatment groups in this study, it may be due to low inclusions rates and warrants further investigation. If the macroalgae A. taxiformis has the potential to perform similarly to the microalgae Schizochytrium spp., this could increase consumers’ preferences for algae-fed beef while also providing producers with a tenderness premium for their product.
The current study showed no statistical differences in consumer sensory evaluation or preference toward steaks from any of the treatment groups in agreement with Kinley et al. (2020) in which steaks from beef cattle fed A. taxiformis were evaluated for taste, tenderness, juiciness, or overall flavor. Another study demonstrated that trained panelists were unable to detect a difference in tenderness in beef steaks from cattle fed up to 150 g of Schizochytrium spp. per day (Phelps et al, 2016). Conversely, some studies reported that the supplementation of Schyzochytrium fed at a 3.89% inclusion rate of DMI in lamb diets and up to 30 g/kg of feed intake to cattle resulted in meat samples as having a “seaweed” or “fishy” flavor in which the authors attributed to increased docosahexaenoic acid levels (Rodriguez-Herrera et. al, 2018; Urrutia et. al., 2016). However, these two studies feed a substantially greater amount of seaweed in their diets than the current study, which could be the reason for the alteration in overall taste. The current study indicates that the supplementation of A. taxiformis at 0.25% or 0.5% to cattle does not significantly impact overall meat quality nor alter the sensory properties of the steaks.
In summary, this study showed that the use of A. taxiformis supplemented to beef cattle diets reduced enteric CH4 emissions for a duration of 21 weeks without any loss in efficacy. The efficacy was highly correlated with the proportion of NDF in the diet. Additionally, A. taxiformis has no measurable residual effect in the product and did not alter meat quality or sensory properties. Importantly, the use of A. taxiformis impacts DMI and not ADG, therefore increasing overall feed efficiency (FCE) in growing beef steers. There may also be potential to reduce the cost of production per kg of weight gain. These feed cost reductions in combination with significantly reduced CH4 emissions have a potential to transform beef production into a more financially and environmentally sustainable and product efficient industry.
ACKNOWLEDGEMENTS
This research received financial support from Elm Innovations, the David and Lucile Packard Foundation and the Grantham Foundation. The authors acknowledge Meat and Livestock Australia, James Cook University and CSIRO for the supply of A. taxiformis used in the trial. We are grateful to undergraduate interns: A. Neveu, A. Wilson, A. Yiao, B. Wong, C. Chow, C. Martinez, C. Mielke, D. Maqueda, E. Anderson, J. DeGuzman, J. Fang, J. Infante, J. Jordan, K. Allchin, K. Garcia, K. Martin, L. Arkangel, M. Cervantes, M. Venegas, M. Zack, P. Nguyen, P. Petschl, S. Calderon, S. Leal, S. Lee, T. Lee, and V. Escobar that participated in the trial. We appreciate Dr. Craig Burnell and Steve Archer (Bigelow Labs in East Boothbay, ME, USA) for developing methods to measure bromoform concentration in Asparagopsis taxiformis, meat, liver, and feces collected in this study.
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