Cattle and other ruminants produce highly digestible human food from materials that are largely inedible by the human population, including grasses, milling residues, oilseed crushing byproducts, etc. This is accomplished through rumen microbial fermentation, which produces usable energy compounds and excellent quality protein. This miraculous anaerobic fermentation process, however, contributes to the production of unwanted greenhouse gases (GHG), in particular methane (CH4) which can trap atmospheric heat.
Many GHG mitigation strategies have been studied, with the goal of reducing GHGs and CH4. While reducing GHG production can be readily accomplished, doing so and maintaining production and feed efficiency has been much more difficult to achieve. Roque et al.  determined that seaweed (Asparagopsis) reduced CH4 production by 67% when fed at 1% of the diet, but this substance also reduced intake and milk production. In the study of van Zijderfeld et al.  nitrate reduced CH4 production by 40% but has the potential to be toxic.
Plant bioactive compounds have been extensively studied, but most lack consistent results, or have unwanted side effects. Tannins, as an example, have been demonstrated to reduce CH4 production, but also bind feed proteins, reducing intestinal digestibility . Goel and Makkar  reviewed saponins as compounds to reduce CH4 production and concluded that these reduce protozoa, but this may or may not translate into reduced GHG. Many essential oil compounds have demonstrated potential in vitro, but limited in vivo testing has been much less promising .
Agolin, an essential oil blend has been shown to alter the rumen biota leading to greater energy efficiency as well as reductions in CH4 and NH3 output. A meta-analysis based on 23 studies demonstrated a 4.1% improvement in energy corrected milk yield after a four-week adaptation period, with no change in dry matter intake. In addition, methane intensity (g/kg ECM) was reduced by 9.9% on average and CH4 output was reduced in all trials in which it was measured . A subsequent feeding experiment  further confirmed the methane intensity reducing potential for Agolin at 11.1%, along with a reduction in NH3 intensity of 16.0%.
The purpose of this study was to evaluate the essential oil blend Agolin in a high producing herd that would be representative of high concentrate feeding and management practices and determine if improvements in yield and efficiency were measurable.
2. Materials and Methods
2.1. Animals and Treatments
Holstein cows from a commercial dairy farm in the Finger Lakes region of New York, USA, were used in this evaluation. The farm housed over 2000 cows in pens of approximately 150 cows/pen. Four pens of multiparous cows were selected for this study based on similarity for average days in milk, and milk production.
Test pens of animals were treated according to normal farm practice. To not impede these practices cows could be moved out of the pens and cows could be moved into the pens, but only cows that remained in the original pens for the full duration of the trial were used in the analysis.
Feed was issued twice daily and consisted of a common total mixed ration (TMR) provided to all 4 test pens. The diet was formulated to meet nutrient requirements using the feed formulation platform NDS (RUM&N Sas, Reggio Emilia, Italy). Cows were given sufficient feed to allow for 2% - 3% orts. Feed issued and orts were measured daily. The essential oil product was added to the TMR through a concentrated farm pack and was calculated to provide 1 g/cow/day.
Milk samples were collected over the last 2 days of the pretrial (May 11 and 12, 2020) and end of the trial (July 18 and 19, 2020) during the regularly scheduled monthly DHIA collection visit. Fresh milk samples obtained by Dairy Herd Improvement Association (DHIA) personnel were sent to the Ithaca New York, USA DHIA Laboratory for the analysis of fat and true protein.
DHIA returned values for milk yield, percentage fat (w/w) and percentage protein (w/w). Component yields were calculated by cow. Fat corrected (FCM) milk and energy corrected milk (ECM) were likewise computed by cow using the equations described by Erdman . Data were analyzed using Minitab 16 statistical software (Minitab Inc., State College PA, USA), the linear model analysis of variance accounting for the effects of treatment, and pen using pretrial values as covariates by cow. Feed intake was analyzed using pen as the experimental unit (n = 4) with days used for replication. Differences were declared significant when the probability (P) of a different result was calculated to be less than 5% (P < 0.05) and were declared a tendency when P was less than 10% (P < 0.10).
3. Results and Discussion
The ingredient composition of the diet used during the study period is given in Table 1. The diet contained 59.6% forage with 70% of the forage as corn silage, with a grain content of approximately 40% of the total dry matter.
The nutrient composition of the diet is shown in Table 2. The composition clearly shows that the diet was a high concentrate diet and would be expected to represent the type of diet typical for high producing dairy cows in the United States.
Pen selection was based on the level of production and average days in milk for all cows available in the pen at the time that the trial was started. While the numbers of animals remained consistent within the pen, some cows were removed, and others were placed in the pens. There were 184 control and 146 test cows that were in the same pens, receiving the correct diets for the full duration of the trial (Table 3). The pre-trial data for these cows showed that they remained well matched across treatments.
Results for milk yield and milk composition were based on cows included in the analysis (Table 4). Milk yield did not change with the inclusion of Agolin, but fat and protein percentages were elevated (P < 0.05). There was a significant improvement in fat yield per cow/day (P < 0.05) resulting in a tendency for FCM and ECM to be greater for the treatment group (P < 0.10). Increased milk fat yield was also found in a California Agolin feeding trial  involving very high producing cows receiving high energy diets.
Efficiency was calculated by pen. There was a tendency (P < 0.10) for both FCM and ECM to be improved when the additive was included in the diet. This is in also agreement with the California Agolin feeding trial .
Table 1. Ingredient composition of the common diet used in the study.
Table 2. Nutrient composition of the common diet used in the study.
Table 3. Comparison of pretrial allocation of animals and final enrollment.
1Values for all cows in pens when pen allocations were made; 2Pretrial values for cows that remained in the pens for the duration of the trial were used in the statistical analysis.
Table 4. Effects of the feed additive Agolin on production parameters in high producing mid-lactation cows1.
1P < 0.05 differences are significantly different: P < 0.10 there is a tendency for differences to be significantly different.
Agolin has been demonstrated to significantly increase FCM and ECM in mid-lactation cows producing more average levels of milk of approximately 30 kg   but prior to conducting the current experiment, it was questioned if the product could improve performance in very high producing cows. During ruminal methanogenesis, CO2 and H+ react to form methane, representing a loss in energy to the host animal. If methanogenesis is inhibited and H+ accumulates, rumen pH declines, and fiber digestion can be reduced. Reducing CH4 production in the rumen without reducing organic matter digestibility requires the H+ to be utilized in the production of volatile fatty acids, mainly propionic acid. This results in the energy being available to the host to use for productive purposes.
Knapp et al.  categorized CH4 mitigation strategies into three categories: 1) Feeding diets to reduce the acetate/propionate ratio, to decrease H+ to be used in the formation of CH4. 2) Improving animal productivity to dilute maintenance and improve feed efficiency. 3) Provide rumen fermentation modifiers. All three strategies were evidently in practice for the test pens of animals as reported here.
This feeding study compared the lactational performances of high producing cows receiving the rumen modifier Agolin® to cows receiving the same diet without the feed additive. The results provide further evidence that Agolin has the potential to increase fat and protein corrected milk in high producing dairy cows. Other trials have also demonstrated the ability of Agolin to reduce enteric methane production. With a combination of improved energy corrected milk and feed efficiency in addition to a decrease in enteric methane emissions Agolin Ruminant shows potential to be of benefit not only to dairy profitability but also to the growing demand for reducing the carbon footprint of dairying.
The authors would like to thank Mr. Doug Young, Spruce Haven Farm, Union Springs, NY, 13160, USA for providing his facilities, expertise and enormous assistance in running this trial. The authors also wish to acknowledge Dr. Essi Evans, E+E Technical Advisory Services, for sorting and compiling data and providing all statistical analyses.
Conflict of Interest
Mr. John Clark and Mr. Peter Williams market Agolin in the United States of America. Both were involved in the design but not the analysis of data or interpretation of results.
 Roque, B.M., Salwen, J.K., Kinley, R. and Kebreab, E. (2019) Inclusion of Asparagopsis armata in Lactating Dairy Cows’ Diet Reduces Enteric Methane Emission by over 50 Percent. Journal of Cleaner Production, 234, 132-138.
 Van Zijderveld, S.M., Gerrits, W.J.J., Dijkstra, J., Newbold, J.R., Hulshof, R.B.A. and Perdok, H.B. (2011) Persistency of Methane Mitigation by Dietary Nitrate Supplementation in Dairy Cows. Journal of Dairy Science, 94, 4028-4038.
 Gerber, P.J., Henderson, B. and Makkar, H.P. (2013) Mitigation of Greenhouse Gas Emissions in Livestock Production: A Review of Technical Options for Non-CO2 Emissions. Food and Agriculture Organization of the United Nations (FAO), Rome.
 Yatoo, M.A., Chaudhary, L.C., Agarwal, N., Chaturvedi, V.B. and Kamra, D.N. (2018) Effect of Feeding a Blend of Essential Oils on Methane Production, Growth, and Nutrient Utilization in Growing Buffaloes. Asian-Australasian Journal of Animal Sciences, 31, 672-676.
 Belanche, A., Newbold, C.J., Morgavi, D.P., Bach, A., Zweifel, B. and Yáñez-Ruiz, D.R. (2020) A Meta-Analysis Describing the Effects of the Essential Oils Blend Agolin Ruminant on Performance, Rumen Fermentation and Methane Emissions in Dairy Cows. Animals, 10, 620-628.
 Carrazco, A.V., Peterson, C.B., Zhao, Y., Pan, Y., McGlone, J.J., DePeters, E.J. and Mitloehner, F.M. (2020) The Impact of Essential Oil Feed Supplementation on Enteric Gas Emissions and Production Parameters from Dairy Cattle. Sustainability, 12, Article No. 10347.
 Erdman, R. (2011) Monitoring Feed Efficiency in Dairy Cows Using Fat-Corrected Milk Per Unit of Dry Matter Intake. Proceeding of Mid-Atlantic Nutrition Conference, College Park MD, 23-24 March, 69-79.
 Santos, M.B., Robinson, P.H., Williams, P. and Losa, R. (2010) Effects of Addition of an Essential Oil Complex to the Diet of Lactating Dairy Cows on Whole Tract Digestion of Nutrients and Productive Performance. Animal Feed Science Technology, 157, 64-71.
 Hart, K.J., Jones, H.G., Waddams, K.E., Worgan, H.J., Zweifel, B. and Newbold, C.J. (2019) An Essential Oil Blend Decreases Methane Emissions and Increases Milk Yield in Dairy Cows. Open Journal of Animal Sciences, 9, 259-267.
 Elcoso, G., Zweifel, B. and Bach, A. (2019) Effects of a Blend of Essential Oils on Milk yield and Feed Efficiency of Lactating Dairy Cows. Applied Animal Science, 35, 304-311.
 Knapp, J.R., Laur, G.L, Vadas, P.A. Weiss, W.P. and Tricarico, J.M. (2014) Invited Review: Enteric Methane in Dairy Cattle Production: Quantifying the Opportunities and Impact of Reducing Emissions. Journal of Dairy Scence, 97, 3231-3261.