Progress report for FNE20-968

Project Type: Farmer
Funds awarded in 2020: $15,000.00
Projected End Date: 05/31/2021
Grant Recipient: Zfarms
Region: Northeast
State: New York
Project Leader:
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Project Information

Summary:

Estimates suggest that sheep account for roughly 8% of global anthropogenic methane emissions, a massive contributor to build-up of greenhouse gases. While methane is far more potent than carbon dioxide, its atmospheric impact declines significantly after only 10 years, which suggests relatively short-term benefits can be achieved by acting now. In this report we seek to refine and extend current research into the use of Asparagopsis taxiformis as a feed additive to reduce methane expulsion by sheep. Our research had three objectives: 1. Assess methane expulsion over a six-week period using 0.0% (control), 0.5%, and 2% Asparagopsis DMI (dry matter intake) levels, 2. Examine changes in the well-being of the three groups using a variety of parasite counts as a proxy, 3. Explore correlation of observed methane and parasite results with sheep microbiome levels and diversity. Consistent with the initial hypothesis, we found 50-70% reduction in methane expulsion when using 2% Asparagopsis DMI, with 93% of the sheep showing improvement after only 3 weeks. However, in a departure with prior studies we found impact at 0.5% DMI dosing more limited. Observed decline in methane for 2% DMI cohort was supported (and partly explained) by statistically significant, 32% decline in Archaea methanogenic organisms. However, our most exciting findings came from analysis of sheep parasites, which we believe is the first time such research was published. We found that use of Asparagopsis had a significant anti-parasitic effect. We observed statistically significant reduction at both 0.5% and 2% dosing levels, with both Eimeria and Strongyles parasites exhibiting declines. We examined both egg count in fecal matter, as well as cultured samples and found confirmation in both approaches. Surprisingly, lower, 0.5%, dose had faster and more significant anti-parasitic benefit. This could have favorable commercial implications given production capacity constraints we are seeing today. It is important to note that given the lack of prior studies, our research was not explicitly structured to test for anti-parasitic properties. Instead, it was found as part of overall sheep health investigation. We were also very encouraged by the fact that treatment groups experienced 1% faster weight gain versus Control. Unfortunately, we were not able to demonstrate link between weight gain and parasite declines – suggesting an alternative mechanism. Examination of microbiome showed statistically significant increase of beneficial Butyrivibrio bacteria for 0.5% cohort. Effect was only marginally significant for the 2% group. We also observed a significant decline in Peptostreptococcaceae bacteria for 2% DMI cohort, which seems to suggest direct antimicrobial impact at higher dose levels​. There was a significant decline in Ruminococcus genera in both intervention groups which might signify a shift to lower Firmicutes to Bacteroidetes ratio and less production of fermentation compounds that might be substrates for methane synthesis. Results to-date are extremely exciting – at least, raising the potential that methane reduction can be achieved, while improving parasite loading and increasing weight gain and beneficial gut bacteria. There are several topics that require further study, with most important being appropriate dosing that balances methane reduction against potentially higher antimicrobial effect. Results also need to be replicated outside of an organic farm, to show efficacy with traditional commercial farming methods.

 
 
 
 

Three groups of investigated animals were :

  1. Control group( 15 Whiteface organic grass fed sheep)(1.5 year olds, non pregnant) – that were not given any seaweed for 6 weeks 
  2. Group 1(15 Whiteface organic grass fed sheep)  (1.5 year olds, non pregnant) were given 0.5% of DMI seaweed a day for 6 weeks
  3. Group 2(15 Whiteface Organic grass fed sheep)(1.5 year olds, non pregnant)   were given 2% of DMI seaweed a day for 6 weeks.
 
 
Project Objectives:

The first goal of the  project is to find an answer if an addition  of red seaweed Asparagopsis taxiformis 0.5%DMI or 2%DMI of organic matter to feed of pasture raised organic sheep would help to control environmentally harmful methane emissions. The second goal was to check supplement’s long term effect on animals health, integrative parasite control,  body score composition and microbial diversity of the intestine in addition to its reduction of methane output and help in mitigating global climate change. 

Introduction:

 

 

Methane CH4 is a greenhouse promoting gas that is a natural product of digestion and fermentation in the stomachs of grazing animals. It is normally produced in the rumen by special methanogenic bacteria that accept extra hydrogen ions formed after fermentation of the cellulose and carbohydrates by fibrolytic and amylolytic bacteria. In this way it protects animals from ruminal PH drop and acidosis that would mean decreased digestive efficiency, decreased activity of rumen enzymes, and danger to overall health. Ruminant animals have the unique ability to digest cellulose and lignin by specialized bacteria in their digestive tract, convert them into short chain fatty acids (propionate, butyrate and acetate), absorb them into the circulation, which is then used for energy balance, growth and metabolism (Ferry, 2011; Macfarlane, 2003). However, excessive production of the methane means that beneficial metabolites are not converted into the Short Chain Fatty acids, and being used for productive energy needs, are diverted into the methane and get excreted. There is an irreversible loss of 2.3% of potentially useful metabolites for every 1 kg of Dry Matter intake, which signifies losses of animal productivity and ultimately decrease in profitability of the farms (Niu et al, 2018). Methane production in the rumen is shown to positively correlate with animal weight, dry matter intake, NDF digestibility, simple sugars and carbohydrates in the diet and negatively correlate with fatty acids intake (Niu et al, 2018). There have been ongoing efforts to reduce methane emission by changing animal feed. Several additives were studied over the years, including nitrates, tannins, saponins, essential fatty acids, ionophores, chemical synthetic bromoform, and vaccinations. Reported methane reduction ranged from 10% to 39% (Williams, 2010; McGinn, 2004; Patra, 2010; Goel, 2012; Cottle,2011). In some studies (particularly with synthetic bromoform) the methane reducing ability gradually declined over time meaning – suggesting potential bacterial adaptation to counteract the trend (Patra, 2017; Belanche, 2019). Several research studies demonstrated that red seaweed Asparagopsis Taxiformis significantly (40 to 90%) decreased methane production in the rumen of the cows, sheep and goats both in vivo and in vitro

(Machado, 2014; Kinley, 2016; Roque, 2019; Machado, 2015; Machado, 2016). The mechanism of methane reducing action of the red seaweed is alteration of the microflora of the rumen with shift of the microbiome into bacterial species producing more hydrogen gas and CO2, instead of CH4. There was no decline of methane reduction over time, but longer-term studies are needed (Machado, 2016). Red algae administration at a 0.5% to 5% DMI was effective at methane reduction, but slightly reduced short fatty acids production for >2% of DMI, and adversely affected organic matter digestibility at doses higher than 5% DMI (Xixi Li, 2016). The supplementation also shifted short fatty acids into higher propionate production. Propionate synthesis serves as an alternative hydrogen acceptor or “sink” and successfully competes with the methane producing pathway in this scenario. Most previous in vivo studies were done on commercial farms where animals were fed forages, plus concentrated feed. It was shown previously that concentrated feed and grains are less digestible than forages, they decrease rumen PH, and result in less cellulolytic hydrolysis and less methane production. There is no agreement on appropriate dosage – in vivo studies point to range between 0.5% and 2% (Roque, 2019); in vitro suggest around 2% (Vijn, 2020). Our study was designed to examine the trade-offs by dosing at 0.5% and 2% DMI. The goal is to achieve sufficient methane reduction, without harming animal health. Parasite infestation is an enormous problem that incurs substantial losses in animal lives and productivity on the farms year after year (Sackett 2006, 2019; Piedrafita, 2010; McRae, 2015). We decided to look at parasite infestation and intestinal microbiota as an indicator of animal overall health and to correlate it with seaweed supplementation at different levels of inclusion and with methane emission.

Parasite control remains a challenge for organic pasture raised sheep and goats that relies on natural substances(herbs,nutritional supplements,minerals, homeopathic preparation) and skillful pasture management. There is a need for a new and innovative solutions for integrative parasite management and control that are  natural, efficient and non toxic to the animals, do not pollute the environment and do not promote antibiotic and anti parasite drugs resistance that are also harmful to human population. 

Asparagopsis taxiformis is a red seaweed that contains large number of different biologically active substances including iodine and bromine halogenated metabolites, total phenolic compounds, flavonoids,chlorophyll, carotenoids,plant sterols,ketones,acetates.  They were found  to have  antibacterial, antiviral, antiprotozoal, antifungal, antioxidant, antiproliferative qualities in human and animal studies

Total phenolic compounds are present as a major molecular group, with an important action in defending against bacteria,wounding and contributing to the antioxidant and free radical scavenging activity in the tissues. The pungent aroma of these algae is due to essential oil composed mainly of bromoform with smaller amounts of other bromine,chlorine, and iodine containing methane, ethane, ethanol,acetaldehyde,acetones, 2-acetohydroxypropanes,propenes,epoxypropanes,acroleins and butenons. 

The pronounced  antibacterial activity against pathogenic shellfish and fish bacteria  relevant to aqua culture-Vibrio cholerae and Vibrio parahaemoliticus was demonstrated in vitro.

A.taxiformis extract produced Quorum Sensisng inhibition of bacterial biofilm.  

The extract of A.taxiformis was active against multiple pathogens of sea bass including P.damsele, Vibrio.harveyi considered to be resistant to most antibiotics in aquaculture when fed by pellets.

A.taxiformis aqueous extract was effective inhibiting  embryonic development of Neobenedenia ectoparasite of infected farmed barramudi fish,reducing hatching success of the parasite to 3% compared to with 99% for the seawater control.

Algal extract has powerful effect on Leishmania infantum achieving 100% mortality of the parasite, and the LD 50 value was 25 mcg/ml for promastigote and 9 mcg/ml for amastigote.

Asparagopsis taxiformis  also contains water soluble polysaccharide carrageenan that is a digestion resistant fiber  that was shown to have prebiotic properties and ability to selectively modulate intestinal microflora with survival of most favorable for the host microorganisms and suppression of the pathogenic microflora. There is an important interaction between bacterial microflora and parasite organisms in the intestine of the vertebrate animals. Alteration of the microflora with the prebiotics and other biologically active substances can make intestine less hospitable to parasites. Favorable microbiota can affect mucus synthesis that can increase expulsion of the parasite, strengthen the permeability of mucus barrier that resists parasite infestation, secrete molecules that inhibit invading parasites, can prime protective immune response by stimulating host Treg cells in the gut.

Our study is done on organic farm and results would need to be confirmed in commercial setting, which utilize concentrated feed.

 

 

 

Description of farm operation:

Z Farms is a certified organic farm in the village of Dover Plains, New York. We conduct certified organic poultry and meat operations and grow berries and vegetables.
The goal is to deliver the freshest possible certified organic and grass fed beef, lamb, goat’s meat, poultry, eggs and also berries and vegetables with the highest nutritional values to local communities. In order to do it we farm responsibly and carefully with the full respect to Earth natural resources and environmental factors.We have our own bees that help pollinate berries and orchard trees. Agriculture that is productive yet replenishing and balancing to the health of the soil and local ecology as a whole is our highest priority.
Crop rotations, cover crops, strip tilling, contour tilling, summer fallow, composting, integrative pest management, biological pest control are just a few examples of our responsible farming practices.
Development of fully sustainable diversified farm operations serving local communities and supplying customers with the best quality fresh meat, poultry, eggs, vegetables, fruits and berries.Our mission: utilization of sound farming and land practices in order to increase soil fertility, increase soil organic matter, build topsoil, minimize fossil fuel usage emphasize environmental stewardship. Animal nutrition is an important component of establishing healthy ecosystem with the human population on its other end.
We have been in operation since 2015.The farm has total 210 acres, out of those 55 acres are actively farmed and 70 acres are protected woods and watersheds. We have 2 full time employees,and 2 part-time workers.We sell direct to consumers at the farmers markets,at the farmstand at the farm and have been running the CSA for vegetables.

Cooperators

Click linked name(s) to expand
  • Alexia Akbay
  • Manigandan Lejeune, Dr

Research

Materials and methods:

The study was approved by Z Farms Organic IACUC (Institutional Animal Care and Use Committee)

Protocol #0012 on May 29, 2020. It was designed in accordance with guidelines set out by USDA Animal

Welfare Act and Guide for the Care and Use of Agricultural Animals by Institute for Laboratory Research.

To gather necessary data, we have conducted a six-week field trial aimed at determining the impact of

additional seaweed feed on sheep methane production, parasite load, and microbiome. Experiment was

designed to evaluate results among 3 groups of 16 randomly assigned 18 months old Whiteface

sheep. First group – Control – served as a baseline and was not given any seaweed. Second group was

given seaweed supplement equal to 0.5% of daily dry matter intake. Group 3 received higher dose equal

to 2% of DMI. Seaweed was administered in feeding troughs and mixed with 20g of alfalfa for

enticement. Levels were based on assumption that a typical sheep consumes roughly 2% of its body

weight on daily basis. Consequently, 0.5% DMI group was given just under 5g/day of seaweed, while 2%

DMI was given 19g/day. We also conducted nutritional forage, alfalfa, and Asparagopsis analysis for

each of the groups (see Table 1). There was no significant difference among groups.

Table 1 – Chemical composition of forage (taken from pasture) for different treatment cohorts, Alfalfa, and Asparagopsis used in the study.

Forage analysis

 Sheep were provided run-in shed for shelter, fresh water, salt and mineral supplements.

Baseline and Testing Chronology

The trial was started on June 1st, while the animals were still on the winter paddocks. At this point, we collected parasite samples which served as a pre-pasture baseline. Samples were collected rectally using sterile lubricated glove, samples were inserted into glass vials, placed on dry ice, and delivered the same day to the Cornell University Animal Health Diagnostic Center for analyses. Immediately following the first sampling, sheep were released on three separate pastures. One week later, on June 8th, we took a second sample of fecal matter for parasite analyses. This was also the point we began to administer Asparagopsis feed supplement. We took two more samples from each sheep three and six weeks thereafter – June 29th and July 21st.

At the same time as we collected samples for parasites, we also took sufficient fecal matter to do microbiome testing. This was inserted into specialty vials, stored, and shipped via same-day delivery (on dry ice) to Weill Cornell Medicine Microbiome Core Lab. We have provided the lab with the set of bacteria and organisms that we wanted to assess as part of the study. These included Archaea, a methanogenic microorganism, as well as variety of bacterium that are deemed important to healthy digestion and healthy microbiome diversity.
Methane testing was done at the start of the trial (June 8th), then at 3 week and 6-week point. In order to do methane testing we built a small enclosure that was connected to corral. Enclosure was designed to minimize air movement while the sheep is being tested, and allow for air clearing between tests (See Exhibit 1).

Each sheep was brought into enclosure for a 3-minute test. Testing was done using Laser Methane, Bluetooth-enabled mini-BE instrument (model # SA3C32A – BE) provided by Pergam Technical Services. Device was linked to Android pad, which was used to store measurement information using a dedicated application from the manufacturer. Settings on the device permitted parsing of the measurement data into 20sec increments, which was averaged within and across those time segments. This mitigated impact of occasional ultra-short spikes in methane, while capturing prolonged emissions. For each sheep we tabulated peak emissions (defined as average over highest 20sec interval), as well as average over the entire 3min period. Results could have some variability in the event the 3min period does not cover a burp or other methane emission. However, we found high consistency in seeing a distinct peak for each of the measurements, which suggested to us presence of an eructation event. Overall use of LMD (laser methane detection) methodology has been well investigated in research and found to have high correlation to other methane measurement techniques.

Finally, we collected several basic measurements that covered weight, body score, and FAMACHA score of each sheep. These data were mostly used to determine existence of any spurious correlation and to monitor health of the animal (make sure they are gaining weight throughout the study).

Statistical Analyses

We have calculated several descriptive statistics for methane, parasite count, and microbiome. In all cases we found that data were highly right skewed and did not feel assumption of normal distribution could be justified. Hence, we looked at a choice of non-parametric test. We selected Wilcoxon signed- rank test to evaluate significance in the change among matched samples. Specifically, whether we could reject the hypothesis that samples came from populations with the same distribution. To calculate relevant statistics we used Excel, as well as an online program provided by Vassar – http://vassarstats.net/wilcoxon.html. Since data was already in Excel, we ran several ANOVA tests and multiple regressions assuming normal distribution, which yielded directionally similar conclusions, but was not included in this report.

 

 

 

 

 

 

Research results and discussion:

This section presents results of our study. Presented analyses are divided into four parts: 1. Weight characteristics, 2. Methane testing, 3. Parasite testing, and 4. Microbiome analyses.

Weight characteristics

At the start of the trial, there was little difference in average weight of the three cohorts (see Table 2). We view weight gains as an indicator of sheep overall health, as well a critical variable for commercial viability. A supplement that reduces methane at a cost of getting a smaller sheep would not be practical. We were pleasantly surprised to see that average gains by groups that were give seaweed were roughly 1% higher, which is a very positive result. Even more reassuring is the difference in the number of sheep gaining weight. As shown at the bottom of the table, Control group had only 56% of the sheep gaining weight, while treatment group had almost 93% experiencing weight gains during the second half of the study. On the surface, this would be supportive of anti-parasitic properties of seaweed that we discuss later in this section. However, we were not able to demonstrate statistical significance by using parasite count, age, and starting weight as explanatory variables. It does not mean that Asparagopsis does not have a beneficial effect, but rather that mechanism may be different from the one we posit. In either case, we are comfortable concluding that use of this supplement has no adverse impact, and likely to be beneficial.

Table 2 – Sheep weight by treatment cohort over the course of the study
Sheep Weight Characteristics of Each Treatment Cohort (lb)

Table 2 Sheep weight in 3 treatment groups and control

 

 

 
 
 

 

Average and median methane emission results for each of the treatment cohorts are shown Table 3, while Table 4 shows average and median for the peak observations. We are encouraged to see relatively similar baseline of methane emissions at the beginning of the study. Furthermore, values that we observed are within range calculated in other studies. Our values are somewhat lower, which we hypothesize may be due to benefit of organic, grass-fed farming.

Table 3 – Average and median Methane Emission (ppm) by treatment cohort – 3min sampling period.

Table 3 Methane emission average and median

 

 

Table 4 – Average and median Peak Methane Emission (ppm) for each treatment cohort. Calculated based on highest 20sec period during 3min sampling.

 

 

Casual examination of average and peak trends suggests a similar behavior. For example, second treatment group (2% DMI) exhibits clear improvement over time, which is evident in both average and median values. Relative to the baseline starting point, improvement at the three-week point is a remarkable 65%, and somewhat lower 50% at the six-week point. Modest increase from week 3 to week 6 is surprising, but there are possible explanations tied to changes in pasture diet and/or some increase in resistance to methane reducing agent. We discuss these possibilities later in the report. We validated our finding using Wilcoxon Signed-rank Test, which showed a high degree of significance at both week 3 and week 6. Statistics are shown in Table 5 below.

Table 5 – Wilcoxon Signed-Rank test for change in methane emissions for 2% DMI treatment group from start of trial to week three and six

Neither control group nor 0.5% DMI group exhibited statistically significant decline in methane emissions. Lack of statistically significant decline for low-dose cohort is surprising and somewhat contradictory to prior studies. This requires a closer look at the underlying data. The most salient characteristic is the difference between average and median values at the end of the study (See Table 2). While average emissions rose by 21%, median declined by 36%, which suggests presence of significant outliers. When we simply examine number of sheep showing improvement in methane expulsion, there is a positive trend (see Exhibit 2).

However, average values for the 0.5% cohort did not improve due to 4 sheep that showed a significant rise in the level of emissions. Given the small sample size any attempt to “exclude the outliers” seems inappropriate. However, we note that all 4 sheep had one common characteristic – significantly higher parasite levels at the beginning of the study. This was evident in both average egg count, as well as growth in culture (i.e. actual hatching). Strongyles parasite concentration was 2x the average of the group. Link between methane and parasites would explain the findings. It has been considered in research and will be discussed later in the report.

Exhibit 2 – Percent of sheep exhibiting a reduction in the methane emission at the end of the six-week study

Parasite testing

We collected data on a several different parasites found in the fecal matter of the sheep. We identified three that existed in sufficient amount to warrant more detailed investigation – Eimeria, Strongyles, and Strongyloides Papillosus. Tabulation of data suggested relatively low, and infrequent occurrence of Strongyloids. Levels were not deemed high enough to significantly affect health of the animal. This was a pleasant surprise since animals are raised organically, without dewormers.

Looking at average (see Exhibit 3) and median (see Exhibit 4) concentration of Eimeria we note a couple of striking trends. First is the remarkable decline between June 1 and June 8, which is the first week sheep were moved from winter paddocks to pasture. This may go a long way toward explaining the need for chemical dewormers and antibiotics for barn-kept animals, relatively to those pasture raised. Declines in parasite concentration continued through the study. However, big difference between average and median values suggests a need for a non-parametric statistical test. We conducted the tests over every periods. Results for parasite reduction were highly significant for the first week across all groups.

Exhibits 3 and 4 – Measurement of Eimeria concentration (eggs/gram) in sheep fecal matter. Results are averaged across sheep for each cohort

However, when examined over the period to treatment (June 9th to July 21st), differences emerge (see

Table 6).

None of the cohorts showed improvements at a three-week mark. Though 2% DMI cohort came close. By week six stark differences emerge. Both cohorts that were given seaweed supplements had statistically significant reduction in Eimeria parasite loading, while control group improved (due to pasturing), but failed statistical significance.

Table 6 – Wilcoxon Signed-Rank test for change in Eimeria parasite count, among different cohorts, at week 3 and week 6 relative to start of the treatment.

We next turned to examination of Strongyles. Given its prevalence among sheep and highly adverse impact on their health and survival, we took a deeper look by examining both initial egg count, cultured count of barber pole worm (Haemonchus contortus), as well as presence of Cooperia.
Qualitatively, trends in Strongyles concentration mimic those of Eimeria (see Table 7). We generally see a sharp drop once the animals are moved from winter paddocks to pasture on June 1. This is followed by mostly steady decline in all groups. Next, we performed statistical analyses, similar with the one used in assessing Eimeria trends (see Table 8).

Table 7 – Average, median Strongyles Haemonchus presence (eggs/gram) in fecal matter for different treatment cohorts.

Table 8 – Wilcoxon Signed-Rank test for change in Strongyles Haemonchus parasite count, among different cohorts, at week 3 and week 6 – relative to start of the treatment.

Just as was the case with Eimeria, we note statistically significant decline in Strongyles concentration by week 6 in both treatment groups, but no improvement in the control group. However, effect seems to be higher for 0.5% DMI cohort in magnitude, and its presence was statistically significant at an earlier, 3- week milestone.

Up to this point we focused solely on presence of parasite eggs in the fecal matter. However, we also

asked the lab to look at the Nematodal larvae culture success rate, which is of greater importance when

assessing health of the sheep. Culturing was done only for the samples that had higher than 100

eggs/gram concentration of Strongyles (Haemonchus contortus) eggs. Culturing below that level was not

deemed useful due to minor impact on overall health. Similarly, we examined emergence of Cooperia

species, where the rise if thought to indicate favorable rebalancing of gut organisms with the shift to less

harmful, and possibly, commensal varieties. Table 9 highlights significant decline in Haemonchus

contortus as treatment continued. In other words, not only are we seeing declines in the egg counts, but

fewer of them hatched. Interestingly, the decline in Haemonchus was particularly notable for the lower

dose group. This is consistent with earlier observation that 0.5% DMI group had earlier and more

significant decline in the Strongyles egg count. Similarly, we are seeing a favorable rise in Cooperia. This

is a very encouraging finding, which suggests that use of Asparagopsis as a feed supplement has not

resulted in higher parasite loads. In fact, we saw a significant decline in both egg count and hatching

propensity. As far as we know, this is the first time such observation was published.

Table 9 – Presence of Haemonchus and Cooperia in cultured samples for different cohorts. Culturing was only done for samples with Strongyles concentration >100 eggs/gram

Microbiome analyses

We analyzed variety of gut bacteria and micro-organisms, using Illumina Shotgun sequencing, that were captured in the fecal matter. Our goal was to assess the impact of seaweed supplement on methanogenic organisms (Archaea), as well as see if there is change in other bacteria. Examining abundance of the Archaea (see Exhibit 6) confirms results of our methane expulsion test. We note a significant decline in the abundance for 2% DMI group, but much more limited impact for control group and 0.5% DMI dosing. These results mimic our conclusions from methane expulsion. Results for the high dose group were statistically significant at 2% level, using Wilcoxon signed rank test.

Exhibit 6 – Changes in Archaea abundance over the course of the study by treatment cohort (%). Data for each sheep is shown as separate point.

We were encouraged to see a statistically significant rise in Butyrivibrio, which is a beneficial gut bacteria, for

0.5% DMI group (see Exhibit 7). Result was directionally similar for higher dose group but did not meet statistical hurdle. There was also statistically significant drop in Ruminococcus for groups

Asparagopsis (see table 10). This confirms one potential pathway for efficacy of the seaweed, as we discuss in the next section. We note a statistically significant drop in Peptostreptococcaceae bacteria for 2% DMI cohort, while drop for other groups was not statistically significant.

Exhibit 7 – Changes in Butyrivibrio abundance over the course of the study by treatment cohort (%). Data for each sheep is shown as separate point.

Table 10 – Average, Median and Statistical Significance of the Drop in Abundance of Ruminococcus Among Treatment Cohorts. Measured in Fecal Matter.

DISCUSSION Methane Reduction

Our results demonstrated significant 60% methane reduction in the exhaled air for pasture-raised organic sheep given red seaweed as a feed supplement at 2% DMI after 3 weeks, and 50% methane reduction after 6 weeks. Mild increase from methane expulsion from 3 weeks to 6 weeks period may be related to somewhat different pasture dry matter intake and composition (mixed grasses, broom grass, clover, alfalfa ratio). Sheep were rotated every week on a fresh green paddock as part of grass-fed operation. Another possibility is that mild increase in average methane production was related to microbial adaptation of the rumen methanogens under selective pressure of red algae, and survival of more anti-methanogen resistant species.

Similar decrement in methane reduction of anti-methanogenic compounds secondary to microbial adaptation was described during administration of synthetic, chemical bromoform to different livestock animals. This rapid decrease in methane reducing efficiency in conjunction with high toxicity precluded use of those compounds on a larger scale in the past (Denman, 2007, Abecia, 2012, Knight, 2011). However, increased resistance to methane reducing effect was not observed for red seaweed algae in the past. Longer than 6 weeks trials are necessary to evaluate this possibility.
Respiration chambers are used to measure CH4 at an individual animal level. Use of chambers is technically demanding, and only a few animals can be monitored at any one time (McGinn et al. 2008). However, these systems provide continuous and accurate data on air composition over an extended period of time. Laser methane detector gave us an opportunity for non-invasive and non-contact scan sampling of enteric CH4. With the possibility for real-time CH4 measurements, the LMD offers a molecular-sensitive technique for enteric CH4 detection in ruminants. Initial studies have demonstrated a relatively strong

agreement between CH4 measurements from the LMD with those recorded in the indirect open-circuit respiration calorimetric chamber (correlation coefficient, r = 0.8, P < 0.001). The LMD has also demonstrated a strong ability to detect periods of high-enteric CH4 concentration (sensitivity = 95%) and the ability to avoid misclassifying periods of low-enteric CH4 concentration (specificity = 79%). Being portable, the LMD enables spot sampling of methane in different locations and production systems (Chagunda, 2013). Our results are in line with a few previously reported investigations of methane reducing properties of Asparagopsis taxiformis in ruminant animals that were done in vivo. Xixi Li (2016) reported significant CH4 emission reduction by 50-80% over a 72-day feeding period with the pelleted grain/straw in Merico sheep supplemented by Asparagopsis taxiformis at approximately 2% DMI. There was no significant effect on methane emission at 0.5% DMI. Breanna Roque (2019) administered Asparagopsis armata which is similar in its biochemical properties to Asparagopsis taxiformis to grain/forage fed lactating Holstein cows at 0.5% DMI and 1% DMI. The significant reduction of methane emission was found at both inclusion rates -26% reduction in methane yield (adjusted for feed intake) at 0.5% DMI and 42% reduction in methane yield for 1% DMI. In contract to Roque, there was no pronounced methane reducing effect in the low 0.5% supplementation group in our trial. One possible explanation is that the sheep in current trial are exclusively fresh forage grass fed and raised only on natural pastures, without additional concentrates and grains added to their ration. It was shown previously that methane production depends on feed composition. More digestible feed (fresh forage as opposed to concentrated grains) leads to formation of more H2 protons available for methane production as result of efficient hydrolysis of plant fibers by cellulolytic bacteria. Addition of grains and concentrated to the ratio decreases rumen PH and makes digestive enzymes less efficient, with the resultant less substrate (H2) available for methane synthesis. Since the sheep in the present study are exclusively raised on forages, it is possible that they produce relatively more methane and need higher doses of anti-methanogenic compounds. Another possibility that 0.5% DMI seaweed inclusion did not produce significant reduction in methane in sheep versus cows might be the genetic differences in rumen microbiology in different species, and even in

 between breeds within the same species. It was shown that rumen metagenome and metatranscriptome of low methane yielding sheep has smaller rumen size and unique Sharpea-enriched microbiome characterized by lactic acid formation and utilization. It results in lower hydrogen production and lower methane formation (Kamke, 2016). It is possible that the White face sheep investigated in this study have relatively larger, and genetically higher, methane producing rumen since animals were bred historically for larger body frames and carcasses for efficient meat production.
There was a significant 1% higher weight gain per sheep in both treatment groups vs control. The sheep were rotated on the pastures for the duration of the experiment so the exact DMI of each individual sheep was not calculated. Based on significantly more weight gain in the supplemented groups, it is assumed that supplemented sheep had improvement in their metabolic parameters and rumen digestion efficiency. Sheep voluntarily consumed an allocated portion of the seaweed mixed with the 20g alfalfa per sheep in the feeding trough every day of the experiment. There was no observable detrimental effect on their behavior or appetite. It is in alignment with the previous investigations conducted in vitro that showed no detrimental effect of Asparagopsis inclusion in the diet at 2% or less of DMI on rumen fermentation efficiency and short chain fatty acids production (Li, 2016).

Microbiome and Potential Mechanism for Methane Reduction

Ruminant animals digest cellulose and lignin from the fibrous material of the pastures and convert them through Embden-Meyerhof-Parnas (EMP) glycolytic pathway to the short chain fatty acids (acetate, propionate and butyrate) with the help of cellulolytic and amylolytic bacteria in the rumen. The SHFA (short chain fatty acids) are absorbed in the circulation from the rumenal wall and are used for energy production, growth and all aspects of metabolism. The common end results of fiber degradation in the rumenare acetate, propionate, butyrate, and less significantly lactate, formate, and H2 -hydrogen. If hydrogen accumulates significantly it can decrease rumen pH and inhibit all enzymatic activity with the

resultant loss of all energy efficiency and metabolism. The common acceptor and neutralizer of elevated Hydrogen in the rumen are methanogenic bacteria from the group archaea that serve as a so-called “hydrogen sink”. They absorb extra hydrogen and combine it with CO2 to produce CH4 that is burped by the animal. Red seaweed works by inhibiting and reducing numbers of methane producing bacteria or archaea, decreasing available “hydrogen sink “and shifting microbial fermentation into more efficient biochemical pathways. The short fatty acid ratio is significantly changed by supplementation with Asparagopsis taxiformis both in vivo and vitro. The production of acetate is decreased, the acetate/propionate ratio is decreased, propionate production is increased after feeding red seaweed. Acetate and butyrateare known to be Hydrogen donors, and propionate serves as hydrogen acceptor in the ruminal glycolytic pathways. The propionate becomes a hydrogen sink instead of methane synthesizing bacteria. It leads to more efficient nutrient utilization, more SCFA production per kg of dry matter and better animal health, since useful fatty acid is synthesized and utilized by the animal using hydrogen molecule instead of horizontal hydrogen transfer to the methanogens and loss of the possible nutrient through methane emission. Total volume of SCFA produced in the rumen is not decreased by Asparagopsis taxiformis supplementation in smaller doses. In a higher amount of supplementation, the SCFA volume gradually declines, which might signify decrease in the animal production and health.

Higher than 5% DMI of supplementation decreases Organic matter digestibility, as well as SCFA synthesis, and precludes its application for agricultural animals, despite total 100% methane emission inhibition (Kinley, 2016).
Our study shows that 2% of DMI is the optimal methane reducing dose for organic fresh forage raised sheep without additional grains or concentrate in their diet. It provided also improved weight gain and body scores. In this way methane reducing strategies accomplish two goals at the same time. One is to reduce methane emission by the animals helping the global ecology, and second – to shift animal metabolism into a more efficient and productive pathway.

The IIlumina shotgun microbiome analysis revealed a significant decrease of Archaea population as well as archaea/bacteria ratio in the feces in both treatment groups compared to control at all treatment points, but only high supplementation group change reached statistical significance. The reduction of Archaea was directly proportional to the reduction of methane emission by the seaweed in the 2% DMI group. It is in alignment with the previous research by Breanna Roque (2019) that showed the average relative abundance of Euryarchaeota (archaea) over the duration of the experiment was lower in A.taxiformis group compared to. control (1.38% and 1.79% respectively). Archaea are methane producing organisms of the rumen and their total content is 3-4% of the total microbial population. They efficiently remove excess hydrogen from the rumen and convert it to methane using the cobamide-dependent methyl transferase step in the release of the CH4. A.taxiformis contains biologically active secondary metabolite bromoform and dibromochloromethane that can react with reduced vitamin B12, and in this way can inhibit critical last step in methane synthesis (Martinez-Fernandez, 2016).

The fecal samples that we collected were analyzed for microbiome abundance and composition in the present study. In the previous research Andrade, 2020, it was shown that there is co-occurrence of Amplicon Sequence Variants of archaea in rumen and fecal microbiome, indicating that fecal Archaea microbiome can be used as a proxy for the ruminal Archaea population in the cattle. Based on those results, we make conclusions on possible rumen archaea physiology analyzing only fecal microbiome in the present study. On the other hand, it is less clear that lower intestinal tract microbiota directly reflects rumen and stomach microbial physiology. There is previous research that shows that bacterial microbiota populates different compartments of the Gi tract independently and there might be no direct correlation in between the species in different parts (Wang, 2017, Tanca,2016). We assumed that there is some correlation between feces microbial composition and upper GI microbiome because of the linear physical nature of the digestive tract and its interconnectivity. Changes in one compartment would cause physiologic and immunologic changes in other areas of the GI tract. This approach obviously has limitations and awaits further confirmation.

Microbiome, Parasite Reduction Mechanism, and Link with Methane

One of the most important indicators of sheep health and nutritional status is the ability to withstand parasite infestation. Hundreds of thousand sheep are lost every year due to uncontrolled parasite sickness, incurring significant economic and financial losses (Mamun, 2020). Emerging parasite resistance against chemical dewormer in commercial operations is a growing problem. Organic pasture raised sheep are particularly vulnerable since they spend all their life cycle on fresh green grasses, which means much higher exposure to the environmental parasites. Chemical dewormers are not allowed in organic production, and operations rely on integrative parasites management including natural herbal compounds, mineral supplements, and particular types of rotational grazing in order to reduce the parasite load.

We used FAMACHA score and fecal egg count to evaluate parasite infestation of the treatment groups sheep and compared them with the control. FAMACHA is a clinical score of the level blood sucking parasites infestation correlating anemia of the animal with the paleness of its lower lid mucous membrane (Gauly, 2004). There was no significant difference in FAMACHA scores among the groups. There was a significant drop in the fecal egg counts in both treatment groups compared with the control. The most important, and deadly, parasite blood sucking nematode Haemonchus contortus from Strongylos sp., decreased sharply in 0.5% DMI supplementation group. The Nematode larval culture success rate was significantly reduced in both treatments as a result of A.Taxiformis supplement. It means that not only fecal egg count declined, but infectious pathogen numbers were diminished. Functional, active parasites from the feces that grew well in laboratory culture were also reduced (Hubert,1984). It signifies that A.taxiformis has substantial anti parasite activity against gastrointestinal nematodes.

Asparagopsis taxiformis contains large number of different biologically active substances including iodine and bromine halogenated metabolites, total phenolic compounds, flavonoids, chlorophyll, carotenoids

plant sterols, ketones, and acetates (Nunes, 2018). They were found to have antibacterial, antiviral, antiprotozoal, antifungal, antioxidant, antiproliferative qualities in human and animal studies (Paul, 2006, Pinteus, 2015). Total phenolic compounds are important in defending against bacteria, wounding and contributing to the antioxidant and free radical scavenging activity in the tissues.

The pungent aroma of these algae is due to essential oil composed mainly of bromoform with smaller amounts of other bromine, chlorine, and iodine containing methane, ethane, ethanol, acetaldehyde, acetones, 2-acetohydroxy propanes, propenes, epoxypropanes, acroleins and butenons (El-Baroty, 2007). Pronounced antibacterial activity against pathogenic shellfish and fish bacteria relevant to aquaculture- Vibrio cholerae and Vibrio parahaemolyticus was demonstrated in vitro (Genovese, 2012). A.taxiformis extract produced Quorum Sensing inhibition of bacterial biofilm (Jha, 2013). The extract of A.taxiformis was active against multiple pathogens of sea bass including those considered to be resistant to most antibiotics in aquaculture (Marino, 2016). A.taxiformis aqueous extract was effective in inhibiting embryonic development of Neobenedeniaecto parasite of infected farmed barramundi fish, reducing hatching success of the parasite to 3% (Hutson, 2012). Algal extract has a powerful effect on Leishmania infantum achieving 100% mortality of the parasite (Vitale, 2015).

Our results showing significant anti parasite activity against Haemonchus Contortus This is the most important pathogen affecting global sheep production. These are new finding and we have not found any indication that they have been explored in the past. There is a growing body of evidence that reduction in greenhouse gas emission is associated with worm control in lambs. Kenyon et al, 2013 showed that clinically parasite infested sheep consistently took longer to reach market weight and generated extra 10% of CO2 per kg of weight gain over healthier non infested sheep, and successful parasite control helped to reverse the trend. Houdjik et al, 2017 demonstrated that parasitism increased calculated global warming potential per kg of lamb weight by 16%, which was similar to the measured impact of parasitism on feed conversion ratio. Fox et al, 2018 showed that the ubiquitous parasite Telagosargia circumcincta drove a 33% increase in methane yield from the infected sheep. The parasitism significantly increased methane

output per kg of DMI, even though DMI was decreased in absolute numbers in sick animals. It signifies that gastrointestinal nematodes in ruminant animals can lead to substantial changes in digestive tract including increased cell turnover, changed permeability, changed PH, altered secretory activity and inhibited gastric acid production.

Parasite infestation has a significant effect on host resident microbial communities. It was shown that Haemonchus Contortus infestation is increasing abomasa PH, negatively affecting digestive function of the sheep, decreasing total amount SCFA produced in the rumen, decreasing DMI, causing diarrhea and weight loss (El-Ashram, 2017). Illumina shotgun microbiome analysis of the feces revealed a significant decrease in the abundance of Ruminococcus species from Firmicutes phylum in both low and high supplementation treatment groups. If fecal Ruminococcus abundance is functionally independent and distinct from rumen microbiome, decrease of its the population after reduction of the parasitism in the rumen might signify the improvement of cellulolytic activity in the rumen with the resultant less fiber available in the large intestine (Andrade, 2020). It explains the subsequent decrease of its abundance in both treatment groups at 3 and 6 weeks. On the other hand, if the interdependence of ruminal and lower intestinal bacteria is assumed, the decrease of Ruminococcus abundance in the feces might directly correlate with rumen bacterial counts. Ruminococcus is one of the most important cellulolytic enzymes of the rumen, digesting fiber with the resultant formation of short chain fatty acids, predominantly acetate, formic acid, H2 and CO2 in the presence of methanogenic bacteria. In this way it can directly contribute to an increase in methane production in the rumen (Tapio, 2017).

When methanogenic bacteria are absent, fermentation is shifted towards succinate and formate, as opposed to acetate and H2, and the amount of methane is significantly decreased (Latham,1977). Because there are less methanogenic archaea available to accept H2 in the rumen secondary to red seaweed inhibition, there is a shift in the Ruminococcus fermentation spectrum to less acetate production and possibly resulting in decrease in the relative abundance of this species. Decrease of Ruminococcus species significantly correlated with the decrease in methane production in the high supplementation

group. Parasite upregulates host carbohydrates and cellulolytic enzymes synthesis as a part of adaptation to the environment and attempts to cause chronic infection where parasites can survive and thrive for a long time (Robert Li, 2015; Leung, 2018; Scotti, 2020). In experimental infection of goats with Haemonchus Contortus, there was a significant shift in the microbiota with the upregulation of biochemical pathways responsible for carbohydrate, amino acid and lipid metabolism (Robert Li, 2015). Since the A.taxiformis might have direct nematicidal effect on the parasite, decrease in parasite infestation might be stimulating less the host’s cellulose digesting microbes, with the resulting decrease in relative abundance of Ruminococcus and all Firmicute species in the rumen with the shift into lower Firmicutes/Bacteroidetes ratio and less resulting total methane production. The diminished drive to increase cellulolytic activity after the reduction of the parasite activity, might be synergistic in shift of microbiome to lower Firmicutes species and possibly more abundant Bacteroides and other non-cellulolytic bacteria (Peachey, 2019). It is a complex interaction between parasite and host microbiota when parasites produce secretory products stimulating more cellulolytic activity of the Ruminococcus species and relative shift in SCFA for more acetate and H2 in the rumen for use to its own metabolic advantage. The result might be an overall increase in methane emissions. Conversely, red seaweed inhibition of both parasites and methanogens, brings methane production down and improves animal health (Peachey, 2019).

Our results showed a significant trend towards increase in Butyrivibrio species particularly in the low supplementation group at 6 weeks of the study. Butyrivibrio genera is a Clostridia species organism from the Firmicutes phylum that as an important physiologic activity in the rumen and all intestinal tract. It degrades polysaccharides and ferments the released monosaccharides to yield short chain fatty acids that are used by the ruminants for production of meat, milk and fiber (Palevich, 2020). The important product of Butyrivibrio bacterias metabolism in the rumen and in the intestine is butyrate that may account for up to 10% of daily SCFA production. It is considered as an important energy source of ruminal and intestinal epithelium, as well as stimulator of the ruminal epithelium growth and function. Butyrate is also known to affect metabolic activity of the ruminal epithelium, abundance of transcripts of proteins mediating SCFA

absorption, epithelial blood flow and rumen motility. It was also shown to exert a trophic and anti- inflammatory effect on stomach and intestinal mucosa (Palevich, 2020).
When infused directly into rumen, butyrate increased efficiency of SCFA absorption from the rumen of the growing goats. In terms of growth performance, butyrate use increased daily weight gain in animals in many studies. Butyrate is a potent inhibitor of inflammation (Gorka, 2017). In a study of Haemonchus Contortus experimentally infected goats, there was a significant difference between uninfected control and H.contortus infected goats (Robert Li, 2015). It is possible that an increase in relative abundance of Butyrivibrio in a low dose treatment group after 3 weeks and 6 weeks was related to epithelial repair and regeneration with the production of anti-inflammatory butyrate after A.taxiformis caused decrease in parasite infestation.

Asparagopsis taxiformis contains a significant amount of polysaccharide carrageenan as one of its biologically active compounds that contains galactose molecules as monomers combined by specific complex carbohydrates 1-4- bonds.(Nunez, 2017). It was shown to have prebiotic qualities in the previous studies.(Han, 2019). Since Butyrivibrio species are able to hydrolyze galactose bonds, red seaweed might serve as a prebiotic for the beneficial intestinal bacteria independent of its effect on ruminal bacteria.

There was a less significant increase in the abundance of the Butyrivibrio in the high treatment group. It is possible that at higher dose A.taxiformis exerted direct antimicrobial effect and inhibited the beneficial bacteria including Butyrivibrio species. The fact that we observed more significant anti parasite effect in low dose group than in high dose group might be a confirmation that the net effect is a sum of direct antiparasitic effect plus shift in the microbiota to more beneficial microbiome communities, that is supportive of hosts improved nutritional status. It is possible that at higher doses, the beneficial effect on the microbial communities is replaced by too strong antibacterial effect, that is less conducive to parasite expelling and restoration of the normal flora.

 

 

Research conclusions:

Conclusions

This study highlights an area of emerging research on ecology and remediation of methane emission by the ruminant livestock animals. The findings demonstrate that at 2%DMI red seaweed Asparagopsis taxiformis reliably decreased methane emission from organic fresh forage fed sheep but not at 0.5%. There was improvement in the productivity, and higher body weight gain in both high and low supplemented sheep. There was a significant decrease in parasite infestation in both supplemented groups, with the low dose group having more prominent effect. Parasite control remains a challenge for organic pasture raised sheep and goats that relies on natural substances (herbs, nutritional supplements, minerals, homeopathic preparation) and skillful pasture management. There is a need for new and innovative solutions for integrative parasite management and control that are natural, efficient and non- toxic to the animals, do not pollute the environment and do not promote antibiotic and anti-parasite drugs resistance that are also harmful to the human population. Asparagopsis taxiformis with its newly discovered anti parasite activity might be a candidate to help manage this long-standing challenge. It is important that methane reducing and antiparasitic effects are investigated in different livestock animals and in different production systems for valid results in future.

Participation Summary
4 Farmers participating in research

Education & Outreach Activities and Participation Summary

2 On-farm demonstrations
1 Online trainings
2 Webinars / talks / presentations
1 Workshop field days

Participation Summary

12 Farmers
1 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Presented the abstract at the North East Integrative Pest Management conference on November,17,2020

https://www.northeastipm.org/about-us/publications/ipm-online-conferences/2020-northeast-ipm-research-update-conference/

Learning Outcomes

20 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation
Key areas in which farmers reported changes in knowledge, attitude, skills and/or awareness:

Integrative parasite control 

Rotational grazing 

Climate change mitigation tools and awareness 

Project Outcomes

5 Farmers changed or adopted a practice
1 Grant applied for that built upon this project
1 New working collaboration
Project outcomes:

The project taught us the importance of intense rotational grazing on parasite control and prevention in organic sheep.

It underscored the importance of integrative parasite control when many different measures(rotational grazing, herbs, supplements, minerals,seaweed) help to improve animals health, prevent losses and increase profits. 

It helped to understand relationship between small farm and planetary ecosystem and livestock contribution into the global  climate mitigation efforts.

Assessment of Project Approach and Areas of Further Study:

The study was well designed. It provided meaningful answer about the efficacy of seaweed supplement for methane reduction. 

The timing might not have been ideal for the demonstration on the parasite reducing effect. The transfer of the animals from the winter paddocks into the fresh green pastures resulted in a large drop in the parasite counts just from the rotations itself. 

It is possibly clouded the anti parasite effect of seaweed supplement. The anti parasite effect might have appeared much more robust if the study was conducted later in summer when the animals had been on the pastures for a longer time and not at the period of transition.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.