TITLE: PLANT-DERIVED FEED SUPPLEMENT FOR REDUCING

METHANE PRODUCTION FROM RUMINANT SPECIES

FIELD OF THE INVENTION

The present invention relates to the field of animal feed and nutrition, especially ruminants, and optimizing the feed of the animals to enhance health, growth, meat and milk production by maximizing nutrients utilization, while decreasing enteric methane emission. A feed supplement consisting of natural substances is disclosed that decreases enteric methane emissions and improves efficiency of nutrient utilization.

BACKGROUND OF THE INVENTION

The domestic animal population has increased by 0.5 to 2.0 percent per year during the last century. One result of this population increase is that gaseous emissions from livestock have become a significant source of atmospheric methane. Domestic animals currently account for about 15 percent of the annual anthropogenic methane emissions.

Methane is produced as a bi-product of normal carbohydrate digestion, especially ruminal fermentation in cattle and other ruminant species. Much of the world's livestock are ruminants—such as sheep, goats, camel, cattle, and buffalo. These animals have a unique, four-chambered stomach. In the forestomachs, bacteria break down feed and generate methane as a by-product. Methane production rate is mainly affected by the quantity and type of feed ingested by the animal and varies among animal species as well as among individuals of the same species.

Ruminant animals are the primary sources of enteric methane. The rumen is filled with microorganisms, billions of bacteria, fungi and protozoa that break down feed into nutrients via fermentative process. The microorganisms process the plant polysaccharides to acetic, propoionic and butyric acids, and gases, methane (CH ₄ ) and carbon dioxide (C0 ₂ ). Ruminants use acetate, propionate, butyrate and the microbial biomass as biosynthetic precursors and sources of energy and amino acids. Hydrogen is an intermediate in the anaerobic breakdown of organic matter in the rumen. It is produced by major fermentative and hydrolytic microorganisms, and then is reutilized by methanogenic archaea that use hydrogen (H ₂ ) to reduced C0 ₂ to produce methane. Some methanogens are associated with the protozoa that reside in the rumen. The majority of the methane is released by belching by the animal, thereby contributing to greenhouse gas increases in the atmosphere.

Methane has 23 times the global warming potential of carbon dioxide. For some nations, animal derived methane production represents greater than 40% of their carbon footprint. The methane produced from enteric fermentation by domesticated livestock is estimated to contribute 25% of US anthropogenic methane emissions (USEPA. 2009. Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990 - 2008). According to the Food and Agriculture Organization (FAO) of the United Nations, about 80 million tons of methane a year enters the atmosphere because of animal digestion. In 2030, it is expected to rise to 128 million tons. Methane contributes most to the global warming impact of milk - according to FAO, about 52% of the total anthropogenic greenhouse gas emissions related to milk production, from both developed and developing countries.

Given the significant impact of livestock on greenhouse gas production, technologic strategies that reduce enteric and manure methane production are desirable (C. Martin, C, D. P. Morgavi, and M. Doreau. 2010, Methane mitigation in ruminants: from microbe to the farm scale. Animal 4:351-365). In the U.S., the dairy industry is taking proactive measures to reduce enteric methane emissions from dairy cattle. The Innovation Center for U.S. Dairy has set a goal of reducing enteric methane missions (per lb. fluid milk) from dairy cattle by 25% by 2020 by implementing the Cow of the Future project (http://www.usdairy.com/Sustainability/^

px). Implementation of existing technologies and management practices in the U.S. dairy industry along with continued genetic progress in milk yields is expected to result in 10 to 12% reductions of methane emissions per unit of milk over the next decade. To achieve the additional 13 to 15% reduction to reach the overall goal of 25% requires investment in research to identify and develop new strategies. Conservative estimates suggest that additional reductions of 15 to 30% can be achieved, dependent upon the development of new strategies through research and their adoption by the U.S. dairy industry (Innovation Center for U.S. Dairy). One of the 8 priority areas of research proposed by the Innovation Center for U.S. Dairy is Rumen Modifiers, which include bioactive compounds capable of reducing ruminal methane production without negatively affecting feed intake, digestibility, and animal performance. Decreasing methane has not only environmental benefits, but a number of carbon offset programs have been initiated which can provide monetary benefits as well. Projects that reduce methane emissions are eligible to earn carbon credits which act as marketable assets. Carbon credits assets are defined by a variety of voluntary specifications, as well as by national and international regulations. Trading occurs on several exchange platforms, including Chicago Climate Exchange and NYMEX Green Exchange.

One carbon credit usually represents the reduction of one metric ton of carbon dioxide or its equivalent in other greenhouse gases such as methane and nitrous oxide. Methane and nitrous oxide have approximately 23 times and 310 times, respectively, the heat-trapping capacity of carbon dioxide. Reducing methane by one ton is equivalent to reducing carbon dioxide by 23 tons. In 2008, about $705 million of carbon offsets were purchased in the voluntary market, representing about 123.4 million metric tons of CC^e reductions. An increasing number of carbon offset programs provide offsets for combustion and containment of methane generated by farm animals. One example is the Chicago Climate Exchange program which allows for agricultural methane emission offsets for methane collection and combustion systems placed into operation in the US, Canada, Mexico and Brazil.

There have been several reports of efforts to decrease methane production and conserving energy for animal growth. In many studies it has been shown that low quality forage is associated with higher methane production, and the converse, higher quality forage, concentrates and protein or energy supplements decrease methane production per unit of feed ingested while enhancing the productivity of the animal, both in weight gain and in milk production. However, there is currently no commercially available product in the form of a supplement or concentrate that has been optimized specifically to address methane emission reduction while enhancing animal performance (Martin et al., 2010).

While some antibiotics and other chemicals, and some plant components have shown inhibitory activity regarding rumen methanogenesis, many have toxic or temporary effects, or decrease feed digestion and consequently animal productivity when deployed in vivo. Thus there is a need for feed supplements that will inhibit methane production in ruminant animals without affecting negatively or enhancing its productivity. SUMMARY OF THE INVENTION

The present invention provides novel plant based feed additives and resultant formulations derived, isolated and/or extracted from Origanum species Also included in the invention are methods for using said additives and formulations by feeding the same to animals to reduce methane emissions from animals.

The current invention is the method and formulation of animal feed supplements comprising Origanum vulgare L., and/or its components, derivatives, and essential oils or other bioactive compounds that singly, or in combined formulations, reduce enteric methane emissions. According to the invention, plant products, including plant tissue, components, and derivatives thereof, including but not limited to essential oils such as carvacrol, geraniol, thymol, limonene, a-pinene, p-cymene, and β-caryophyllene as well as other components such as : di- and tri-terpenoids, lipids and fatty acids, phenols, phenolic and hydroxycinnamic acids, quinones, and flavonoids are added to feed in an amount effective to reduce methane gas emissions from a ruminant animal.

The invention also comprises methods of use of the feed supplements by providing the same to a ruminant animal to that the feed supplements are ingested. The feed supplement compounds of the present invention may be contained in dried leaves or other plant parts from plants with such compounds or taken as purified and/or synthesized preparations.

DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of Origanum vulgare L. (OV) on methane production in vitro (Example 1). Data are means ± SE. Blank = 0 mg/L OV, monensin = 5 mg/L (final medium concentration) monensin (Sigma Chemical Co.), OVL1 = 312.5 mg/L OV, OVL2 = 1,250 mg/L OV, OVL3 = 2,500 mg/L OV, OVL4 = 5,000 mg/L OV. Blank vs.

monensin, P = 0.21; blank vs. OVL1, P = 0.27; blank vs. OVL2, P = 0.23; blank vs.

OVL3, P = 0.07; blank vs. OVL4, P = 0.003 (SE = 8.0 to 41.3).

Figure 2 shows the effect of Origanum vulgare L. (OV) supplementation (500 g/d) on rumen methane production rate in dairy cows (mean ± SE; n = 50; effect of treatment, P = 0.004, effect of time, P = 0.02, treatment * time interaction, P = 0.05).

Figure 3 shows in vitro methane-suppressing effect of specific compounds from oregano essential oil. Figure 4 shows denaturing gradient gel electrophoresis profiles of methanogens produced from community DNA samples from whole ruminal contents from cows fed control or Origanum vulgare L. (OV; 500 g/d) supplemented diets. The lanes are labeled with the cow number, period number, group number, and treatment. The calculated similarity coefficients determined using Bionumerics software are shown on the top left- hand side (n = 12).

DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless otherwise defined, the technical, scientific and medical terminology used herein has the same meaning as understood by those informed of the art to which this invention belongs. However, for the purposes of establishing support for various terms that are used in the present application, the following technical comments, definitions and review are provided for reference.

"Global warming" is the increase in the average temperature of the Earth's near- surface air and oceans in recent decades and its projected continuation.

"Greenhouse gases" are components of the atmosphere that contribute to the greenhouse effect. Without the greenhouse effect the Earth would be uninhabitable. In its absence, the mean temperature of the earth would be about 19°C (2°F) rather than the present mean temperature of about 15°C (59° F). Greenhouse gases include in the order of relative abundance water vapor, carbon dioxide, methane, nitrous oxide, and ozone.

"Carbon dioxide" (chemical formula: CO2) is a chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom. It is a gas at standard temperature and pressure and exists in Earth's atmosphere in this state. Carbon dioxide is an important greenhouse gas because it transmits visible light but absorbs strongly in the infrared.

"Methane" is a chemical compound with the molecular formula CH ₄ . Methane is a relatively potent greenhouse gas with a high global warming potential (i.e., warming effect compared to carbon dioxide).

The "rumen" forms the larger part of the reticulorumen, which are the first two chambers in the alimentary canal of ruminant animals. It serves as the primary site for microbial fermentation of ingested feed, enabling ruminants to eat fibrous plants that monogastric animals cannot digest. "Methanogens" are archaea that produce methane as a metabolic byproduct in anoxic conditions such as the rumen.

"Ozone" (O3) is a triatomic molecule, consisting of three oxygen atoms. It is an allotrope of oxygen that is much less stable than the diatomic O2. It can be used for bleaching substances and for killing microorganisms in air, on surfaces, and in liquids.

"Ruminants" refers to any hoofed animal of the suborder Ruminantia and the order Artiodactyla, characteristically digesting its food in two steps. Ruminants include cattle (both dairy and beef), sheep, goats, llamas, giraffes, bisons, camels, buffalo, deer, elk, wildebeest, antelope, pronghorn, alpacas and yaks.

"Feed efficiency" is a term used to describe the efficiency with which animals convert ingested feed into usable products, such as meat and milk. Feed efficiency is critically important in modern animal agriculture as animal systems that more efficiently convert feed into milk and meat utilize valuable nutrient resources more efficiently, have a lesser environmental impact, and are financially more profitable.

"Fat-corrected milk" is a term used to equalize milk with different fat content. Milk produced by lactating dairy cows varies in its fat content and to compare data among studies or farms, the raw milk yield is converted to fat-corrected milk yield, usually 3.5% fat or 4.0% fat-corrected milk.

"Milk off flavor" Good quality milk should have a pleasantly sweet and clean flavor with no distinct aftertaste. Because of the perishability of milk and the nature of milk production and handling procedures, the development of off-flavors/odors is not uncommon. Off-flavors can be absorbed, bacterial, or chemical in origin. Consumers will reject milk with off-flavors and any feed supplement for lactating cows causing off-flavor will have no market acceptance.

"In vitro experiments" are experiments carried out outside of the animal, i.e. in test tubes in the laboratory. In the case of testing compounds for reducing ruminal methane emissions, in vitro tests are conducted so the investigator is able to screen for positive effects a large number of compounds, which would not be possible with live animals. Many compounds may have methane-inhibitory effects, but most of them are also detrimental to the ruminal fermentation by inhibiting useful microorganisms and thus reducing overall feed digestibility and animal productivity. It is critically important to account for these detrimental effects when selecting bioactive compounds for anti- methanogenic effects.

By "administer", is meant the action of introducing oregano according to the invention into the animal's gastro-intestinal tract. More particularly, this administration is an administration by oral route. This administration can in particular be carried out by supplementing the feed ration intended for the animal with oregano, the thus supplemented feed ration then being ingested by the animal. The administration can also be carried out using a stomach tube or any other means making it possible to directly introduce the same into various parts of the animal's gastro-intestinal tract.

By "effective amount", is meant a quantity of Origanum vulgare and/or its components and derivatives thereof, including essential oils such as carvacrol, geraniol, thymol, limonene, a-pinene, p-cymene, and β-caryophyllene as well as other components such as : di- and tri-terpenoids, lipids and fatty acids, phenols, phenolic and

hydroxycinnamic acids, quinones, and fiavonoids sufficient to allow improvement of feed efficiency, milk production milk nitrogen efficiency, and less resultant methane emission. This effective amount can be administered to said ruminant animal in one or more doses.

The invention consists of a method for supplementing animal feed with the plant components of the invention at concentrations suitable for feed supplements from plants. "Concentrations suitable as feed supplement" means that compounds in amounts of dried plant material or liquid obtained from fresh plants that make up a daily or twice daily dose of an effective concentration of the plant compounds. The concentration may be optimized as to components of the plant shown to have the desired anti-methane activity.

The current invention is the method and formulation of animal feed supplements comprising Origanum speces, particularly, Organum vulgare L., and/or its components, derivatives, and essential oils that singly, and particularly in combined formulations, reduce enteric methane emissions. According to the invention, plant products, including plant tissue, components, and derivatives thereof, including essential oils such as carvacrol, geraniol, thymol, limonene, a-pinene, p-cymene, and β-caryophyllene as well as other components such as : di- and tri-terpenoids, lipids and fatty acids, phenols, phenolic and hydroxycinnamic acids, quinones, and fiavonoids are added to feed in an amount effective to reduce methane gas emissions from a ruminant animal. In one embodiment an animal is fed an amount of oregano plant material form about 100 g /cow per day to about 750 per cow per day. On a per animal body weight basis (kg), this amounts to about 0.15g/kg to about 1.25 g/kg of body weight. Animals can be fed this amount for any period of time as the oregano was found to have no deleterious effects on the animals, but typically they should be fed for a period of at least 7 days, but even a single day can make a difference in ruminal fermentation and animal performance. In another embodiment, the essential oil comprising primarily carvacrol is administered to the animal in an amount of from about 400 mg/L to about 2,000 mg/L of rumen contents.

The Origanum genus comprises several different species, the most common of which is Oregano (Origanum vulgare L.), which is part of the lamiaceae or mint family. The most important species are O. vulgare (pan-European) O. onites (Greece Asia Minor) and O. heracleoticum (Italy, Balkan peninsula, West Asia). For use in the present invention, any of the species of the Genus Origanum is contemplated, for use in the invention, this includes the following species and any and all subspecies within the same, Origanum boissieri, Origanum calcaratum, Origanum cordifolium, Origanum dictamnus , Origanum saccatum, Origanum solymicum, Origanum symes, Origanum akhdarense, Origanum cyrenaicum, Origanum hypercifolium, Origanum libanoticum, Origanum pampaninii, Origanum scabrum, Origanum sipyleum, Origanum vetteri, Origanum acutidens, Origanum bargyli, Origanum brevidens, Origanum hausskenchtii, Origanum husnucan-baserii, Origanum leptocladus, Origanum rotundifolium, Origanum amanum, Origanum bilgeri, Origanum micranthum, Origanum microphyllum, Origanum

minutiflorum, Origanum majorana, Origanum onites, Origanum syriacum, Origanum dayi, Origanum isthmicum, Origanum jordanicum, Origanum petraeum, Origanum pinonense, Origanum ramonense, Origanum elongatum, Origanum floribundum, Origanum grosii, Origanum vulgare, Origanum compactum, Origanum ehrenbergii and Origanum laevigatum.

The main constituents of oregano are its essential oil fraction (maximum 4% of the leaves) which may contain variable amounts of phenols, carvacrol and thymol. Herein, "essential oil" means an oil derived by any means from a plant source. Additionally, a variety of monoterpene hydrocarbons, such as limonene, di- and tri-terpenoids, ocimene, caryophyllene, beta-bisabolene and p-cymene, as well as monoterpene alcohols, such as linalool and 4-terpineol, have been reported to be part of the essential oil derived from the oregano plant. Oregano has also been found to have other compounds including lipids and fatty acids, phenols, phenolic and hydroxycinnamic acids, quinones, and flavonoids that may be used according to the invention. The invention includes not only the entire oregano plant, but also various components of the plant which may be derived or purified therefrom and which, when administered in an effective amount, reduce methane emissions from the animals.

The plant derived compounds may be administered as ground/powdered leaves and stems of plants in which they naturally occur, or in pelleted form (powder combined with binder, such as molasses or glycerol). The compounds may also be administered as a liquid, ingested directly or sprayed upon feed, or added to plant materials subsequently pelleted. Alternatively the compounds may be administered as purified or chemically synthesized compounds in an acceptable carrier. The compounds of the present invention are intended to be administered orally in the form of powder or pelleted feed supplements, suspensions, solutions, pastes, gels, boluses or other suitable means for ingestion. Other compounds may be added to prolong to sustain levels of desired components within the rumen to enhance anti-methane activity or to increase rumen propionate levels or otherwise assist in achieving the desired effects.

According to the invention, the feed for a ruminant animal which can be supplemented is typically selected from feeds containing fibers and/or cereals. Examples of feeds according to the invention are grass, various hays, alfalfa, straw, grains, oilseeds, or any other type of fodder used for feeding herbivores, but also any type of granulated feed, in particular based on wheat bran, oat husks, alfalfa, barley, corn, fruit pomace, cane molasses, low-grade rice flour, straw, soya oil cake, etc.; or also other types of vegetables, such as carrots.

A dietary supplement of the present invention can have a varied combination of biologically active ingredients, such as amino acid and/or between about 0.1-30 weight percent of one or more active ingredients selected from vitamins, trace elements, proteins, non-protein nitrogen compounds, medicaments, enzymes, and the like.

Vitamins either singly or in combination include thiamine HC1, riboflavin, pyridoxine HC1, niacin, biotin, folic acid, ascorbic acid, vitamin B. sub.12, vitamin A acetate, vitamin K, vitamin D, vitamin E, and the like.

Trace elements include compounds of cobalt, copper, manganese, iron, zinc, tin, iodine, vanadium, selenium, and the like. Protein ingredients are obtained from sources such as dried blood or meat meal, cottonseed meal, soy meal, dehydrated alfalfa, dried and sterilized animal and poultry manure, fish meal, powdered eggs, canola meal, and the like.

Protein equivalent ingredients include urea, biuret, ammonium phosphate, and the like.

Medicament ingredients either singly or in combination include promazine hydrochloride, chlorotetracycline, sulfamethazine, monensin, poloxalene, and the like. Oxytetracycline is a preferred antibiotic for cattle prophylaxis. Enzymes of choice include lipolytic proteins which aid feed digestability, e.g., by hydrolysis of fatty acid glycerides to free fatty acid and glycerol.

A feedstock for ruminants such as lactating cattle normally will include silage, and energy concentrate and protein concentrate of the following types: Typically, the feed is supplemented with said feed supplement so that the animal receives an effective amount of bacteria to improve the digestibility and assimilability of the fibres and/or cereals contained in the animal's feed.

By "supplementing", within the meaning of the invention, is meant the action of incorporating the effective amount of oregano or its derivatives according to the invention directly into the feed intended for the animal. Thus, the animal, when feeding, ingests the oregano according to the invention which can then act to reduce concomitant methane emission.

Thus, another subject of the invention relates to a feed supplement for a ruminant animal comprising at least one component of oregano and a carrier.

Carriers

Compositions of the invention may, but need not include a carrier. Carriers used in compositions of the invention can function to give desired characteristics to the compositions of the invention. Examples of such desired characteristics include but are not limited to flowability of the composition, texture of the composition, or stability of the composition.

Any materials that can impart these desired characteristics, and are not detrimental to the animal may be utilized if desired, as carriers in compositions of the invention.

Suitable carriers for making a feed additive composition known in the art including, but not limited to, rice hulls, wheat middlings, a polysaccharide (e.g., specific starches), a monosaccharide, mineral oil, vegetable fat, hydrogenated lipids, calcium carbonate, gelatin, skim milk powder, phytate and other phytate-containing compounds, a base mix, and the like. A base mix typically comprises most of the ingredients, including vitamins and minerals, of a final feed mixture except for the feed blend (e.g., commeal and soybean meal).

In one embodiment of the invention materials that can be used as carriers include but are not limited to cornstarch and silicon dioxide.

In one embodiment of a composition of the invention, the carrier includes both com starch and silicon dioxide. In this embodiment, the silicon dioxide is about 5 wt% to about 20 wt% of the weight of the carrier and the com starch is about 80 wt% to about 95 wt% of the weight of the carrier. In another embodiment, the silicon dioxide is about 7.5 wt% to about 17.5 wt% of the weight of the carrier and the com starch is about 82.5 wt% to about 92.5 wt% of the weight of the carrier.

In yet another embodiment, the silicon dioxide is about 10 wt% to about 1 wt% of the total weight of the carrier, and the com starch is about 85 wt% to about 90 wt% of the carrier. In a further embodiment, the silicon dioxide is about 14 wt%, and the com starch is about 86 wt% of the carrier (also illustrated as the silicon dioxide being about 5 wt% of the total weight of the composition and the com starch being about 30 wt% of the total weight of the composition

In accordance with one embodiment of the invention the foodstuff is fed in combination with the oregano or its derivatives to any mminant animal (i.e., animals with a four-chambered complex stomach). Ruminant animals that can be fed a foodstuff according to the invention include agricultural animals include cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghom, and nilgai.

Any animal feed blend known in the art can be used in accordance with the present invention such as rapeseed/canola meal, cottonseed meal, soybean meal, and commeal, but soybean meal, canola meal, and commeal are particularly preferred. The animal feed blend is supplemented with the oregano, but other ingredients can optionally be added to the animal feed blend. Optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides and polysaccharides. Optional amino acid ingredients that can be added to the feed blend are arginine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HC1, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HC1, and analogs, and salts thereof. Vitamins that can be optionally added are thiamine HC1, riboflavin, pyridoxine HC1, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. Minerals, protein ingredients, including protein obtained from meat meal or fish meal, liquid or powdered egg, fish solubles, whey protein concentrate, oils (e.g., soybean, rapeseed or canola oils), cornstarch, calcium, inorganic phosphate, copper sulfate, salt, and limestone can also be added. Any medicament ingredients known in the art can be added to the animal feed blend such as antibiotics.

The feed compositions can also contain enzymes such as proteases, cellulases, xylanases, and acid phosphatases.

Antioxidants can also be added to the foodstuff, such as an animal feed

composition. Oxidation can be prevented by the introduction of naturally-occurring antioxidants, such as beta-carotene, vitamin E, vitamin C, and tocopherol or of synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, tertiary- butylhydroquinone, propyl gallate or ethoxyquin to the foodstuff. Compounds which act synergistically with antioxidants can also be added such as ascorbic acid, citric acid, and phosphoric acid. The amount of antioxidants incorporated in this manner depends on requirements such as product formulation, shipping conditions, packaging methods, and desired shelf-life

Buffering Agents

In one embodiment of the invention a buffering agent can enhance the digestive functioning of the animal and can aid in counteracting the effects of heat stress.

Compositions of the invention may also include at least one buffering agent.

Buffering agents function to buffer the stomachs of the animals that consume the buffering agent. In cows, a buffering agent functions to aid in digestion of fiber in the cow's diet. A dairy cow has a complex acid-base regulatory system with the pH of the rumen generally varying from about 5.5 to 6.8. If the pH of the rumen is not optimal, microbial yield and efficiency drops, dry matter intake declines, and metabolic disorders can increase. In one embodiment, a rumen buffering agent ties up hydrogen ions near the desired rumen pH.

In one embodiment of the invention, the buffering agent used is sodium

bicarbonate.

The following examples will illustrate the practice of the present invention in further detail. It will be readily understood by those skilled in the art that the following methods, formulations, and compositions of novel compounds of the present invention, as generally described and illustrated in the Examples herein, are to be viewed as exemplary of the principles of the present invention, and not as restrictive to a particular structure or process for implementing those principles. Thus, the following more detailed description of the presently preferred embodiments of the methods, techniques, formulations and compositions of the present invention, as represented in the Examples, is not intended to limit the scope of the invention, as claimed, but is merely representative of presently preferred embodiments of the invention.

EXAMPLES

Example 1

A. Laboratory, in vitro experiments:

A total of 88 essential oils and 14 herbs, including Origanum vulgare L., were evaluated in a batch culture in vitro screening experiments as potential anti-methanogenic additives for ruminant diets. Rumen inoculum was obtained from a lactating dairy cow. Incubation was conducted in serum bottles containing 1 g of a feed mixture (containing essential oils or herbs), 20 ml of buffer, and 20 ml of ruminal inoculum. Incubation was at 39°C for 6 h. Herb inclusion level was from 312.5 to 5,000 mg/L incubation medium (in the case of oregano, LI through L4 on Figure 1) of plant material. Blanks and monensin were also incubated. Monensin is an ionophoric antibiotic commonly used to regulate ruminal fermentation in beef and dairy cattle. In this study, it was used as a positive control at 5 mg/L, final medium concentration. At the end of the incubations, total gas and methane production were measured. As Figure 1 shows, compared with the blank (and monensin), oregano {Origanum vulgare L.) decreased methane production by 55% (160 vs. 350 μg methane, respectively) at the highest inclusion level (P = 0.003) and tended to decrease it at the 2,500 mg inclusion level (P = 0.07). The 5,000 mg/L inclusion level would correspond to 500 g/d oregano supplementation of the diet of a mature dairy cow.

B. Lactating dairy cow Experiment 1:

Based on results from the in vitro experiment (A), a lactating cow trial was conducted to study the effects of dietary addition of oregano (Origanum vulgare L.) on methane production in the rumen and cow performance. Eight primiparious and multiparious Holstein cows (6 of which were ruminally cannulated) were used in a switch over design with two, 21 -d periods. Cows received 500 g/d grass hay (control) or 500 g/d oregano in addition to their basal diet. Cows averaged 41 ± 3.3 kg/d milk yield and 80 ± 12.5 days in milk at the beginning of the trial. Intake of dry matter averaged 26 ± 0.83 kg/d and did not differ (P = 0.24) between treatments. Average milk yield, (43.6 and 44.1 kg/d, control and oregano, respectively; P = 0.61), milk fat, protein, and lactose concentrations were unaffected by diet. Absolute milk nitrogen secretion was not affected by treatment, but as proportion of nitrogen intake (i.e., milk nitrogen efficiency), was greater (P = 0.04) for OV compared with the control. Milk sensory parameters were not affected by treatment (P = 0.33). Fat-corrected (3.5%) milk yield and 3.5% fat-corrected milk feed efficiency were increased (P = 0.03 and P < 0.001) by the oregano treatment compared with the control (42.2 vs. 40.7 kg/d and 1.63 vs. 1.53 kg/kg, respectively). Rumen methane production was measured using the sulfur hexafiuoride (SF ₆ ) tracer gas technique before and 2, 4, 6, and 8 h after feeding. Across sampling points, the oregano treatment resulted in 40% lower (451 vs. 745 g methane/d; P = 0.004) rumen methane production compared with the control (Figure 2). The conclusion of this experiment was that oregano plant material fed at 500 g/d to high-producing dairy cows reduced methane production in the rumen by 40% during the sampling period, which resulted in increased 3.5% fat-corrected milk yield and feed efficiency. C. Lactating dairy cow experiment 2

Lactating cow experiment 1 was repeated with graded levels of Origanum vulgare L. As with Exp. 1, this experiment investigated the effects of dietary supplementation with Origanum vulgare L. leaves (OV) on ruminal methane production, milk production, and milk fatty acid composition in dairy cows. The experimental design was a replicated 4 x 4 Latin square with 8 ruminally-cannulated Holstein cows (DIM, 160 ± 38) and 20-d experimental periods. Treatments were: control (no OL supplementation), 250 g cow/d OV (LOR), 500 g/d OV (MOR), and 750 g/d OV (HOR). The oregano leaves were supplemented to the basal TMR replacing an alfalfa-cottonseed hulls mix. The basal TMR (14.7% CP, 36.2% NDF) contained 58% forage (DM basis; corn silage, alfalfa haylage, and a grass-straw mix). Rumen methane production was measured using the sulfur hexafluoride (SFe) tracer gas technique before and 2, 4, 6, and 8 h after feeding. Methane production tended to be reduced (P = 0.076) by all oregano treatments (16.5, 1 1.7, and 13.6 g kg DMI, LOR, MOR, and HOR, respectively) compared with the control (18.2 g/kg DMI). Oregano supplementation linearly decreased (P = 0.02; SEM = 1.80) DMI: 28.3, 28.3, 27.5, and 26.7 kg/d, control, LOR, MOR, and HOR, respectively. Milk yield tended to increase (P = 0.07; SEM = 7.43) quadratically with OL supplementation: 43.4, 45.2, 44.1, and 43.4 kg/d, respectively. As a result, feed efficiency was increased (P < 0.001) for all OV-supplemented diets (1.59, 1.60, and 1.63 kg/kg, respectively) compared with the control (1.46 kg/kg). Milk protein and lactose concentrations and yields and fat-corrected milk yield were not different among diets. Milk fat content tended to be increased (P = 0.07) by HOR compared with the control (3.57 vs. 3.26%, respectively), but OV tended to linearly reduce (P = 0.07) milk fat. Milk urea-nitrogen concentrations were lower (P = 0.04) for all OV diets (7.95 to 8.49 mg/dL) compared with the control (9.26 mg/dL).

Rumen fermentation (pH, volatile fatty acids, and ammonia concentrations) was not affected by treatment. Apparent total tract digestibility of nutrients was also not affected by OV, except neutral-detergent fiber digestibility was slightly decreased (P = 0.04) by all OV diets compared with the control (49.3 vs. 51.3%, respectively). Oregano had no effect on milk fatty acid composition. In this short-term study, oregano leaves fed at 250 to 750 g/d decreased linearly DMI and tended to quadratically increase milk yield in dairy cows. Feed efficiency was increased with all OV inclusion levels. Oregano leaves tended to reduce methane production in the rumen during the sampling period but had no other effects on ruminal fermentation Milk FA composition was not affected by OV.

D. Summary of the animal experiments:

A summary of the two lactating dairy cow experiments is presented in Table 1. In Exp. 1 (8 cows, 6 of which were ruminally-cannulated, in a cross-over design), 500 g/cow/d oregano supplementation increased, compared with the control, 3.5% fat-corrected milk yield and fat-corrected milk feed efficiency. Ruminal methane production per unit of dry matter intake was decreased 40% by the oregano treatment within the 8 h sampling period. Oregano supplementation had no effect on milk taste. A 48 -participants sensory panel determined that milk from cows fed oregano was not different (P = 0.33) from the control. In a follow-up experiment with 8 ruminally-cannulated cows (4 x 4 Latin square design), milk yield tended to increase quadratically with oregano supplemented to the diet at 250, 500, and 750 g/cow/d compared with the control. Feed efficiency was linearly increased by all oregano treatments compared with the control. Ruminal methane production per kg of dry matter intake tended to be lower during the sampling period (by 10 to 36%; P = 0.076) for the oregano treatments, compared with the control.

Table 1. Effect of Origanum vulgare L. leaf material on milk production, feed efficiency, and enteric methane production in dairy cows (from Tekippe et al., 2010 and Hristov et al., 201 la)

Oregano supplementation,

Item 0 (control) 250 500 750 SEM P-value

Experiment 1

Milk yield, kg/d ¹ 41.0 - 42.5 - 4.86 0.03

Feed efficiency, kg/kg ¹ 1.54 - 1.64 - 0.03 <0.001

Methane production, 31.1 - 18.8 - 9.87 0.007 g/kg dry matter intake/d

Experiment 2

Milk yield, kg/d 43.4 45.2 44.1 43.4 7.43 0.072 ²

Feed efficiency, kg/kg 1.46 1.59 1.60 1.63 0.19 0.004 ³

Methane production, 18.2 16.5 11.7 13.6 2.30 0.076 ³ g/kg dry matter intake/d

¹ 3.5% fat-corrected milk yield and feed efficiency.

Quadratic effect of oregano supplementation.

³ Control vs. oregano-supplemented diets. E. Identification of bioactive compounds responsible for the methane-suppression effect of oregano:

To identify specific compounds responsible for the methane-reduction effect of oregano in the rumen, the chemical ingredients of Origanum vulgare L. were studied. Sixteen compounds most predominant in the plant and essential oil of oregano were identified and screened for methane-suppressing effect in an in vitro system similar to the one described under A. Application levels varied from 400 to 4,000 mg/L ruminal inoculum. Several compounds effectively inhibited methane production (Figure 3) and are likely responsible for the in vitro and in vivo anti-methanogenic effect of Origanum vulgare L. These compounds are: carvacrol, geraniol, and thymol.

Further experimentation is planned to: (1) repeat the animal experiments and test the effect of varying levels of oregano on ruminal methane production and performance of high-producing dairy cows; (2) test the effect of carvacrol, geraniol, and thymol on methanogenesis in animal trials; and (3) investigate the methane-inhibitory effect of other bioactive compounds from oregano.

Example 2

Rumen Fermentation and Production Effects of Origanum vulgare L. in Lactating

Dairy Cows

A lactating cow trial was conducted to study the effects of dietary addition of oregano leaf material (Origanum vulgare L.; 0, control vs. 500 g/d, OV) on ruminal fermentation and methane production, total tract digestibility, manure gas emissions, N metabolism, organoleptic characteristics of milk, and dairy cow performance. Eight primiparious and multiparious Holstein cows (6 of which were ruminally cannulated) were used in a switch-over design trial with 2, 21 -d periods. Cows were fed once daily. The OV material was top-dressed and mixed with a portion of the total mixed ration (TMR). Cows averaged 41 ± 3.3 kg/d milk and 80 ± 12.5 days in milk (DIM) at the beginning of the trial. Rumen pH, concentration of total and individual VFA, and microbial protein outflow were not affected by treatment. Ruminal ammonia N concentration was increased by OV compared with the control (5.3 vs. 4.3 mmol/L). Rumen methane production was reduced 40% in the OV treatment (P = 0.004). Intake of DM (average of 26.6 ± 0.83 kg/d) and apparent total tract digestibly of nutrients did not differ between treatments. Average milk yield, milk fat, protein, and lactose concentrations, milk urea nitrogen, and somatic cell count were unaffected by diet. Milk sensory parameters were also not affected by treatment. Fat-corrected (3 g/kg) milk yield and fat corrected milk feed efficiency were increased (P = 0.03 and < 0.001, respectively) by OV compared with the control (42.2 vs. 40.7 kg/d and 1.63 vs. 1.53 kg/kg, respectively). Urinary and fecal N losses, and manure ammonia and methane emissions were unaffected by treatment. Under the current experimental conditions, supplementation of dairy cow diets with 500 g/d of Origanum vulgare L. had no effect on nutrient intake and digestibility, but drastically reduced methane production in the rumen within the 8 h sampling period. Cows fed Origanum vulgare L. produced more fat-corrected milk and had increased feed efficiency.

MATERIALS AND METHODS

Animals and treatments

The experiment was conducted under the approval of The Pennsylvania State University Animal Care and Use Committee (IACUC# 29566) and involved 4 multipara and 4 primiparous Holstein dairy cows averaging 41 ± 2.6 kg/d milk yield, 2.1 ± 0.78 lactations, and 80 ±10 DIM at the beginning of the trial. The OV used in the experiment was grown in Greece and purchased from Ameriherb Inc. (Ames, IA). Six of the 8 cows were fitted with soft plastic ruminal cannulae (10.2 cm internal diameter; Bar Diamond Inc, Parma, ID). Treatments were arranged in a crossover design with 2, 21-d periods. Each treatment group contained 4 cows including 3 ruminally-cannulated. Cows were balanced across treatments based on DIM, current milk yield, and parity. Control cows received a basal TMR (Table 1) top-dressed with 500 g/d of chopped fescue grass hay (Festuca arundinacea). Experimental cows received TMR top-dressed with 500 g/d of OV. The fescue hay and OV were hand-mixed with a portion of the TMR during feeding. Although no specific measurements were collected, it was observed that cows consumed all OV offered. Cows were housed in a tie-stall facility and were fed once daily at approximately 0800 h. Feed was pushed up 4 to 6 times daily. The basal diet was balanced to meet or exceed NRC (2001) requirements for lactating dairy cows weighing 640 kg and producing 44 kg/d milk with 3.20% fat and 2.93% true protein. At the actual DMI of the cows, the basal diet exceeded the requirements for NE _L , but was slightly deficient in MP (-67 g/d for the control, or about 3%). All diets were fed ad libitum to achieve approximately 5% refusals. Cows had access to fresh water throughout the duration of the trial. During the trial, cows were milked daily at 0530 and 1730 h and received rBST at 14 day intervals (Posilac, Elanco Co., Greenfield, IN; 500 mg, i.m.) beginning at 63 DIM.

Sampling and analyses

Feed intake was measured daily and TMR and refusals samples were collected twice weekly and composited by week. Total mixed ration and refusals samples were dried for 48 h at 65°C in a forced-air oven and then ground in a Wiley Mill (A.H Thomas, Philadelphia, PA) through a 1-mm screen for chemical analyses. All TMR and refusals samples were then composited by period. Aliquot samples were pulverized at 30 hz/s for 2 min using Mixer Mill MM 200 (Retsch, Haan, Germany) for N analysis. Individual forages were sampled weekly and dried in a microwave oven to determine DM. Weekly changes were made to the TMR, if changes occurred in forage DM.

Rumen samples were collected on 2 consecutive days during week 3 of each experimental period. Whole ruminal contents were collected at 0, 2, 4, 6, and 8 h after feeding from the ventral sac, atrium, and 2 samples from the feed mat. After mixing, an aliquot of the whole ruminal contents was frozen at -20°C for bacterial, archaeal, and fungal profile analyses. The remaining sample was filtered through two layers of cheesecloth and the filtrate was immediately placed on ice for further analyses.

Eleven spot urine (approximately 300 mL each) and fecal (approximately 400 g each) samples were collected from each cow at 0900, 1500, and 2100 h on d 2; 0500, 1100, and 1700 h on d 3; 0000, 0700, 1300, and 1900 h on d 4; and at 0200 h on d 5 of week 3 of each experimental period. Urine samples were collected by massaging the vulva. Fecal samples were collected from the rectum or ground, if fresh. Urine was acidified (pH < 3) using 2M H2SO4; pH was verified using litmus paper. Acidified urine was diluted 1 :10 with distilled water and stored frozen at -20°C. Fecal samples were oven dried at 65 °C and composited per cow and period on an equal DM basis. Aliquots of fresh urine

(unacidified) and feces were composited on a wet basis by cow and period and frozen immediately at -20°C for later analysis of ammonia and methane emitting potential of manure.

Body weight was recorded for 3 consecutive days at the beginning and end of the trial and at the start of the second period. Cows were weighed using the Afifarm 3.04E scale system (S.A.E. Afikim, Rehovot, Israel) while exiting the milk parlor.

Milk production was recorded daily while samples for milk composition were collected 3 times during week 3 of each experimental period. Morning and evening samples were analyzed separately so milk component concentration and yield could be weighed for a.m. and p.m. milk yields. Samples were preserved using 2-bromo-2- nitropropane- 1 ,3 diol. Entire milk yield was collected one consecutive night and morning milkings to be composited by treatment and used for milk sensory panel evaluation.

Blood samples were collected from the coccygeal tail vein or artery 2 h after feeding. Blood samples were collected on 2 consecutive days during week 3 of each trial period. Approximately 3 mL of blood was collected into an evacuated tube (Becton Dickinson, Franklin Lakes, NJ) containing 15 mg of sodium fluoride and 12 mg of potassium oxalate. At the same time approximately 5 mL of blood was collected into an evacuated tube (Becton Dickinson, Franklin Lakes, NJ) containing 0.79 mg of sodium heparin. All tubes were placed immediately in ice and, within 1 h, centrifuged at 4°C at 1,500 x g for 15 min. Plasma was recovered and frozen at -20°C for future analyses. Rumen cheesecloth filtrates were immediately analyzed for pH (pH meter 59000-60 pH Tester, Cole-Parmer Instrument Company, Vernon Hills, Illinois) and processed for analyses of ammonia (Chancey and Marbach, 1962), VFA (Y ang and Varga, 1989), total free amino acids (TFAA; ninhydrin procedure, Snell and Snell, 1954), and protozoal counts. Samples for protozoal enumeration were preserved (Hristov et al., 2001) and counted according to standard procedures (Dehority, 1993) using the Sedgewick-Rafter chamber (Hausser Scientific, Horsham, PA).

The effect of OV on ruminal microbial populations was analyzed using 2 procedures: denaturing gradient gel electrophoresis (DGGE) analysis (Hristov et al., 2009) and tag-encoded FLX amplicon pyrosequencing (bTEFAP) using Gray28F

5'GAGTTTGATCNTGGCTCAG and Gray 19r 5' GTNTTACNGCGGCKGCTG primers (Dowd et al., 2008a,b). Fungal tag-encoded FLX amplicon pyrosequence (fTEFAP) was performed utilizing ITS1-ITS4 fungal primers, ITS1-F

'CTTGGTC ATTTAGAGGAAGT AA ITS4R 5' TCCTCCGCTTATTGATATGC. Whole rumen contents samples were composited per cows and period and used for these analyses. For the DGGE procedure, DNA was extracted using the RBB+C method (Yu and

Morrison, 2004); PCR thermoprofiles, DGGE conditions, and cluster analysis of the banding patterns were as explained before (Hristov et al., 2009). Tag-encoded FLX amplicon pyrosequencing analyses utilized Roche 454 FLX instrument with titanium reagents, titanium procedures, a one-step PCR, mixture of Hot Start and HotStar high fidelity taq polymerases, and amplicons originating and extending from the 28F for bacterial diversity and ITSl for fungal diversity. The bTEFAP pyrosequencing were based upon titanium protocols (Roche, Indianapolis, FN) and procedures were performed at the Research and Testing Laboratory (Lubbock, TX) based upon RTL protocols

(www.researchandtesting.com).

Methane production in the rumen was measured utilizing the sulfur hexafluoride (SF ₆ ) tracer technique (Johnson et al., 1994). The SF ₆ permeation tubes were purchased from Dr. Keith Lassey (National Institute of Water and Atmospheric Research, Wellington, NZ). The tubes had an average SF ₆ release rate of 3.04 ± 0.131 mg/d, were placed in the reticulum of the cows on d 1 of the experiment, and remained there throughout the duration of the study. Rumen gas samples were collected directly from the rumen through a modified rumen cannulae (for description see Hristov et al., 2009) placed in 3 h prior to gas collection. Gas samples were collected at 0, 2, 4, 6, and 8 h after feeding during one day of week 3 of each experimental period. A 60-mL syringe was used to remove 110 mL of rumen gas into a 100-mL vacuumed serum bottle. Bottles were refrigerated until analyzed for methane and SF ₆ using gas-liquid chromatography (Hristov et al., 2009). Production of methane was calculated as the release rate of SFg times the ratio of the concentration of methane to SF ₆ in the ruminal headspace (Johnson et al., 1994).

Ammonia, methane, and nitrous oxide emitting potential of manure were analyzed in a steady-state gas emission system (Wheeler et al., 2007) as described by Hristov et al. (2009). Gases were analyzed on a photoacoustic gas analyzer (IN OVA Model 1412; AirTech Instruments, Ballerup, Denmark).

Dry matter intake was calculated by adjusting daily as-fed feed intake to DM content (measured for 48 h at 65°C) of the weekly diet and refusals composited samples. Diet, refusals, and fecal composited samples were analyzed for amylase-treated NDF (aNDF) and ADF using the Ankom ²⁰⁰ Fiber Analyzer (Ankom Technology, Macedon, New York) according to Van Soest et al. (1991) with heat stable amylase (Ankom

Technology, Macedon, NY) and sodium sulfite (Fisher Scientific, Waltham, MA) used in the aNDF procedure. Nitrogen content for diet, refusals, fecal, and urine were analyzed on a CostechECS 4010 C/N/S elemental analyzer (Costech Analytical Technologies, Inc., Valencia, CA) and multiplied by 6.25 to obtain CP values. Total nonstructural carbohydrates (TNC) were analyzed on fecal and diet samples that were reground to pass through a 0.5-mm screen, and analyzed using a procedure described by Smith (1981) with the modification to use potassium ferriccyanide as a colorimetric indicator.

Apparent total tract digestibility of nutrients was estimated using indigestible NDF as an intrinsic digestibility marker (Foley et al., 2006). Indigestible NDF in feed and fecal samples was analyzed according to Huhtanen et al. (1994), with the exception that 25-μηι pore size Ankom filter bags were used for the rumen incubation. Diet, refusals, and fecal samples were ashed for 4 h at 600°C for analysis of OM.

Blood plasma samples were analyzed for glucose (Sigma Glucose Kit 510, Sigma Chemical Co., St. Louis, MO) and plasma urea N (PUN; Stanbio Urea Nitrogen Kit 580, Stanbio laboratory, Inc., San Antonio, TX).

Urinary purine derivatives (allantoin and uric acid) excretion was used to estimate duodenal microbial N flow (for equations see Hristov et al., 2009). A ratio of purine N to total N in rumen microorganisms of 0.134 was assumed based on the data of Valadares et al. (1999).

Milk samples were analyzed for fat, true protein, lactose, and MU (Pennsylvania DHIA, University Park, PA) using infrared spectroscopic method (method 927.16; AO AC, 2005; MilkoScan 4000; Foss Electric, Hillerod, Denmark). Organoleptic properties of milk were evaluated by a 48-subjects sensory panel. A balanced reference duo-trio test was used to determine if the OV milk was significantly different from the control milk.

Panelists were presented with 3 cups: 2 controls and 1 OV milk sample labeled with 3 digit blinding codes and a labeled reference, which was either the control or OV sample. The presentation order was counterbalanced with reference being presented in the left position and panelists were instructed to identify the coded sample that was the same as the reference. Water and saltine crackers were provided as palate cleansers. Evaluations took place in individual testing booths using Compusense ® five software (release 4.6, Guelph, Canada).

Representative samples of OV (250 g of dried material in 3 replicates) were steam distilled for 60 min in 2-L Clevenger type distillation units as described elsewhere

(Zheljazkov et al, 2008). Extracted EO was analyzed by GC-MS on a Varian CP-3800 GC coupled to a Varian Saturn 2000 MS/MS (Palo Alto, CA) and methods of Kovats (1965) and Adams (2009). The GC was equipped with a DB-5 fused silica capillary column (30 m x 0.25 mm, film thickness of 0.25 μηι). The injector temperature was 240°C and the column temperature was initiated at 60°C, increased at 3°C/min to 240°C, and held for 5 min. The carrier gas was He and the injection volume was 1 (10: 1 split). The MS conditions were mass range of 40 to 650 m/z, filament delay of 3 min, target TIC of 20,000, pre-scan ionization time of 100 μβεΰ, ion trap temperature of 150°C, manifold temperature of 60°C, and a transfer line temperature of 170°C. Essential oil constituents were each identified by first determining their Kovats Index using previously reported methods (Kovats, 1965) and comparison of mass spectrum with that reported by Adams (2009). Final confirmation was accomplished by comparison of retention times and mass spectra data with authentic standards when needed. Carbon 13 NMR data was used in the confirmation of carvacrol due to the Kovats Index and mass spectrum similarities with thymol. Essential oil constituents were quantified by performing area percentage calculations based on the total ion chromatogram combined area. For example, the area for each reported peak was divided by the total integrated area from the total ion

chromatogram from all reported peaks and multiplied by 100 to arrive at a percentage. The reported percentage is a percentage by weight in the extracted EO.

For quantification of antioxidant capacity of OV, the ORAC assay was used (Prior et al, 2003). Three samples of approximately 250 mg of OV leaves were weighed and extracted with hexane by sonication, twice. The hexane fractions were combined and used to quantify ORAC of the lipophilic fraction (ORACii _po ). The same samples, after hexane extraction, were then extracted with 70% acetone (30% water; v/v) acidified with 0.5% glacial acetic acid, with sonication for 20 min, twice. The fractions were combined and analyzed for hydrophilic components of the antioxidant capacity (ORAC _hydro ). Samples were analyzed with 4 (ORAChydro) or 3 (ORACii _po ) replicates and quantified using a standard curve (using trolox, a vitamin E water-soluble equivalent) ran in the same plate. The results are expressed in μηιοΐ of Trolox equivalents (TE)/g of dry plant material. Statistical analysis

Data was analyzed using the PROC MIXED procedure of SAS (2003; SAS Inst. In., Cary, NC). Intake, digestibility, rumen TFAA and microbial data, urinary excretions, and milk composition were analyzed assuming a crossover design. The 3 milk

composition samples collected during each experimental period were averaged per cow, and the average values were used in the statistical analysis and to calculate FCM, milk NE _L , and milk fat, protein, and lactose yields. The model used was:

Yijki = μ + gi + c(g)ij + P _k + Ti + e _ijk i Where: μ is the overall mean, gi is the group, c(g)i _j is the cow within the group, P _k is the k _fh period, Ti is the 1 _t treatment, with the error term assumed to be normally distributed with mean = 0 and constant variance. Group and cow within group were random effects and all others were fixed.

Ruminal pH, ammonia, VFA concentrations, and methane production rate data and milk yield were analyzed as repeated measures assuming a crossover design and an ar(l) covariance structure. The model used was: Yi _jk i _m = μ + gi + c(g)i _j + P _k + Ti + D _m + TDi _m + e _ijklm

Where: μ is the overall mean, g; is the group, c(g)y is the cow within the group, P _k is the k _th period, Ti is the 1 _th treatment, D _m is the time effect, TDi _m is the treatment x time of sampling interaction with the error term e^i _m assumed to be normally distributed with mean = 0 and constant variance. Group and cow within group were random effects and all others were fixed. Only significant interactions are published and discussed.

Cumulative ammonia and methane emissions from manure data were fitted (PROC NLIN, SAS) to a quadratic model: (a + b x time + c x time ² ; adjusted R ² was > 0.99 for all datasets). Estimated overall emission lines were compared between treatments using the dummy variable regression technique (PROC NLMIXED, SAS; Bates and Watts, 1988).

Statistical differences were declared at < 0.05. Differences between treatments at 0.05 < P < 0.10 were considered as a trend toward significance. RESULTS

Diet ingredient changes were not made throughout this trial and forages remained the same. Chemical composition of OV and the fescue hay is shown in Table 2. The fescue hay had higher concentrations of fiber and lower CP and NFC than OV. At 26 kg/d DMI and 500 g/d supplement inclusion rate, however, this difference would have little impact on ruminal fermentation or cow performance.

The main compound of the oregano EO was carvacrol (Table 3). Carvacrol represented 91% of the EO, which classifies the OV used in this study as carvacrol-type, based on Baser (2002). Gama-terpinene is commonly found in oregano EO (Baser, 2002) and represented around 2.3% in this EO. Thymol and linalool (a major terpene in oregano EO) were not detected.

Oregano leaves used in this study had a total ORAC (ORACTAC) of 2,082 μ ^η ιοΐ TE/g of dry plant material. The ORACTAC is the sum of the ORACii _po plus the hydrophilic ORAC (ORAChydro). Although specific antioxidant components of oregano were not analyzed, the lipophilic fraction usually contains EO components, vitamin E and A, and other fat soluble components. The average ORACii _po was 41 μηιοΐ TE/g, while the average ORAC _hydro (usually containing flavonoids and other polar components) was 2,041 μηιοΐ TE/g. Treatment had no effect on rumen pH, concentrations of total and individual VFA, TFAA, and protozoa counts (Table 4). There was only a trend for increased acetate to propionate ratio (P = 0.10) for cows supplemented with OV. Ammonia N concentration was increased (P < 0.001) in the OV treatment. No treatment by time interactions were observed for pH, ammonia, or total and individual VFA. The average methane production in the rumen was decreased 40% (average over the sampling period; P = 0.004) over the 8 h sampling period by OV compared with the control. There was a treatment by time interaction for methane production rate (P < 0.05; Figure 2). Except for the initial sample (2 h) after feeding, average methane production was consistently lower for OV than the control throughout the sampling period.

Results from the DGGE analysis are shown in Figure 4. The samples tended to cluster mostly by experimental period and treatment had no effect on the DGGE banding patterns. Bacteroidacea and Clostridiales were predominant bacteria in the rumen, constituting more than 35% of the total bacterial population (based on the pyrosequencing analysis; Table 5). Clostridium spp. were increased (P = 0.003) and Bacteroidacea were decreased (P = 0.03; although Bacteroides were increased) by OV compared with the control. Species such as Ruminococcus albus and Prevotella ruminicola, considered typical ruminal bacteria were detected in low levels. No significant differences were observed in the archaeal populations. Methanobacteriaceae were the predominant methanogens in these samples constituting around 60% of all archaea. Neocallimastix spp. were the predominant fungi (data not shown) and treatment had no effect on their proportion of the total fungal population in the rumen (27 and 23%, control and OV, respectively; P = 0.71).

Intake of DM (Table 6) and dietary nutrients (data not shown) were not affected by treatment. Apparent total tract digestibility of DM (63%), OM (64%), CP (59%), aNDF (34%), ADF (29%) and TNC (91 %) were also not affected by OV (P = 0.62 to 0.98; data not shown). Cow BW, milk yield, milk NE _L output, and milk protein, lactose, and MUN concentrations were not affected by treatment. There was a numerical trend (P = 0.09 and 0.11, respectively) for increased milk fat and feed efficiency for the OV-supplemented cows. Milk fat yield, 3.5% FCM yield, and 3.5% FCM feed efficiency were increased (P = 0.03 to < 0.001) for cows fed OV compared with the control.

Participants in the milk sensory panel were asked to identify which coded milk sample was the same as the presented reference milk under the duo-trio test design. Of the 48 total responses collected, 31 correct responses were required to establish significance at a = 0.05. Sensory results showed that milk from cows fed OV was not different from the control, as it received only 26 correct responses (P = 0.33; n = 48).

Urinary excretions of allantoin and uric acid were not affected by treatment (Table 7). As a result, estimated microbial N flow from the rumen was also not different between treatments. Bacterial purine:total N ratio was not analyzed in this experiment. As the basal TMR was identical for the 2 diets and there were no significant differences in ruminal fermentation (apart from the effect on ammonia concentration and methane production), we assumed that the purine:total N ratio in ruminal bacteria would be similar between treatments in this experiment. In some cases, contrastingly different diets (barley- vs. corn-based) produced similar bacterial purine:total N ratios (Hristov et al., 2005).

Urinary and urea N excretions tended to be decreased by OV compared with the control. Absolute and relative (to intake) fecal and total N losses were unaffected by treatment. Absolute milk N secretion was also not affected by treatment, but as proportion of N intake (i.e., milk N efficiency), was greater (P = 0.04) for OV compared with the control.

Concentration of PUN was increased (P = 0.05) by OV compared with the control. Plasma glucose concentrations were similar between treatments (data not shown).

The ammonia and methane emitting potentials of manure were not affected by treatment (P = 0.76 and P = 0.67, respectively; data not shown). Nitrous oxide

concentration in manure gas was negligible (around 0.6 mg/m ³ ).

DISCUSSION

The OV material used in the current experiment was selected based on in vitro incubations of EO and plant materials (Tekippe et al., 2010). In this in vitro batch culture screening study, herbs and EO were evaluated as potential anti-methano genie additives for ruminant diets. A total of 88 EO and 14 herbs were tested. Origanum vulgar e L. leaf material was among the most effective anti-methanogenic additives and had no adverse effect on VFA production and in vitro NDF digestibility. In this screening experiment 4 levels of OV were tested; the highest level, 5,000 mg/L, reduced (P = 0.003) methane production by about 54% compared with the blank. Compared with the blank, all levels of OV increased (P < 0.001) the 24-h NDF digestibility (24.8 vs. 27.6 to 30.0%, respectively). Therefore, the 5,000 mg/L OV application level, which would correspond to 500 g OV/cow per day for a 100-L rumen volume (assuming all OV was immediately consumed), was chosen for the current experiment.

Overall, the reported effects of EO and plant products on ruminal fermentation have been very inconsistent, which is not surprising given the large variability in products, active compounds, application levels, and experimental conditions (Benchaar et al., 2009). A general trend of decreasing ammonia concentration, but inconsistent effect on ruminal VFA appears from reviewing published studies (Benchaar et al., 2009). At higher doses, most EO products act as antimicrobials and inhibit ruminal fermentation, decreasing ammonia and VFA concentrations (Busquet et al., 2006; Benchaar et al., 2009).

This is the first study in which effects of Origanum vulgar e L. have been evaluated in lactating dairy cows. Larger doses of oregano EO (300 and 3,000 mg/L) clearly inhibited ruminal fermentation in vitro (Busquet et al., 2006). A trend for increased acetate to propionate ratio and decreased propionate concentration, similar to the present experiment, was also observed in vitro by Cardozo et al. (2004) when supplementing 15 mg/kg DM of oregano EO. This may be attributed to the low molecular weight of carvacrol and thymol, which have been shown to permeate and disrupt the cell membrane of gram positive bacteria that produce propionate (Sivropoulou et al., 1996). Wang et al. (2009) reported a significant increase in total VFA concentration, but no substantial changes in molar VFA proportions, in sheep receiving a commercial oregano EO product (Ropadiar).

The increase in ammonia N concentration observed in this experiment is somewhat surprising as in most studies, EO have reduced ruminal ammonia concentration (Benchaar et al., 2009). Ammonia concentration was consistently higher for OV throughout the sampling period (data not shown), which is in line with the increased PUN concentration in the OV cows. Processes such as deamination of feed amino acids, microbial lysis, absorption, and microbial uptake regulate ammonia concentration in the rumen (Hristov et al., 2005). The increased ammonia concentration observed with OV in this experiment could be attributed to the presence of carvacrol in oregano EO. Busquet et al. (2005a) reported in vitro that carvacrol decreased large peptide concentrations and increased ammonia N concentrations 2 h after feeding. Despite of the increased PUN, however, OV cows tended to have reduced urinary N losses, which may indicate a better N utilization post-ruminally. This and the combination of numerically lower N intake and numerically greater milk yield resulted in greater efficiency of conversion of dietary N into milk protein for the OV treatment.

The reduction in ruminal methane production with OV was large in this study and although in line with our in vitro results (Tekippe et al., 2010), was in discrepancy with the ruminal fermentation and microbial profiles data. Most of the hydrogen gas formed during carbohydrate fermentation in the rumen is utilized by the methanogens to reduce carbon dioxide to methane (Wolin et al., 1997). Disposal of hydrogen is considered critical for efficient rumen fermentation and as propionate is an alternative to hydrogen electron receptor, there is a strong inverse correlation between propionate formation and methane production in the rumen (Janssen, 2010). In the current experiment, it is not clear if propionate production and rate of absorption were affected as a result of the reduction in methane production by OV, although propionate concentration was not different between treatments. The relationship between methane production and rumen VFA is not always clear, even in a relatively simple to balance in vitro system. Busquet et al. (2005b), for example, reported a 75% reduction in methane concentration (batch culture experiment) with 300 mg/L garlic oil. At the same time the absolute propionate production was reduced; although concentration was slightly increased, total VFA concentration was about 20% lower with the garlic oil treatment. A review of the effect of EO on ruminal fermentation did not find a clear relationship between rumen propionate and methane production (Hart et al., 2008). In other experiments, however, there was a good agreement between propionate and methane production (Watanabe et al., 2010). Hydrogen concentrations were not measured in the current experiment, but accumulation of hydrogen in the rumen cannot be excluded as a consequence of the reduced methane production with OV (Garcia-Lopez et al., 1996; Kung et al, 2003). As pointed out by Janssen (2010), the processes of methane reduction and hydrogen accumulation are interrelated in the rumen. Thus, hypothetically it is possible that both increased propionate production and absorption and hydrogen accumulation occurred with the OV treatment in the current experiment. Information on these processed, however, was not collected.

The SF ₆ tracer method has been criticized for producing larger variability than established techniques for measuring methane production, such as respiration chambers. Clark (2010), for example, found a good agreement in group means methane emission measurements between SF ₆ and the chamber methods. Variability, however, was about twice as large for the SF ₆ method and the correlation between emission values obtained from individual animals and repeatability in the estimated rates was low (Clark, 2010; Pinares-Patino et al., 2010). Indeed, the variability in our methane production data was large (Figure 2). This large variability may be partially due to variable methane emission rates following feeding (for most cows emissions increased after feeding), but was probably also a result of variability due to the SFg technique. Various factors may have affected methane emission measurements in this study. Cecile et al. (2010), for example, reported lower emission estimates with lower SF ₆ release rate tubes. The SF ₆ tracer may be retained within the digestive tract (Lassey et al., 2010), although this may be affecting less ruminal headspace than breath sampling sites, and may behave differently than the trace gases (Pinares-Patino et al, 2010). Extended sample collection periods may help reduce this variability (Gere et al., 2010; Lassay et al., 2010). The original SF ₆ method was developed for measuring enteric methane production in intact animals as an alternative to the chamber technique, where animals may behave differently than in their natural environment, which may affect enteric gas production rates (Johnson et al., 1994). Our approach in this and other studies (Hristov et al., 2009, for example) was to sample the ruminal headspace, assuming these samples would accurately represent eructated gas composition. There are very few studies comparing rumen headspace vs. breath sampling. Cecile et al. (2010) found similar methane emission rates between breath and ruminal headspace sampling methods using the SF ₆ technique. In contrast, Coates et al. (2010) found the SF ₆ procedure unsuitable for measuring enteric gas emissions from ruminally- cannulated cattle. These authors, however, did not sample the rumen headspace directly, as in the current study, which undoubtedly had affected their conclusions. The SF ₆ permeation tubes were left in the reticulo-rumen throughout the duration of the current experiment, which could have affected SF ₆ release rates. Indeed, emissions were lower (P = 0.006) in period 2 vs. period 1 of the trial (19.3 vs. 30.3 g/h, respectively; SEM = 8.21). However, there was no period x treatment interaction (P = 0.50) for the methane data. The reason for these reduced rates in period 2 cannot be determined, but it apparently did not affect our comparative results and overall conclusions. Nevertheless, due to the large variability in methane emissions within cow during the sampling cycle and between cows on the same treatment, the effect of OV on methane production reported here has to be interpreted with caution. In addition, our measurements covered only a period of 8 h after feeding. We did not collect information on methane production during the remaining 16 h of the feeding cycle. Thus, we cannot be certain that the effect of OV persisted during the entire 24 h until the next day feeding. Others have also reported antimethanogenic effect of oregano EO. Wang et al. (2009), for example, observed about 12% reduction in methane production in the rumen of sheep treated with a commercial product based on oregano EO.

It is likely that the effect of OV on methnogenesis observed in vitro by Tekippe et al. (2010) and in the current study is due to a combination of compounds in the oregano plant. Apart from volatile EO, oregano contains many bioactive compounds such as di- and tri-terpenoids, lipids and fatty acids, phenols, phenolic and hydroxycinnamic acids, quinones, and particularly flavonoids (Skoula and Harborne, 2002). Many of these compounds have been shown to have antimicrobial properties (Baricevic and Bartol, 2002) and may also suppress ruminal archaea. The high antioxidant capacity of the oregano leaves used in this study supports this possibility. For example, the ORAC _TAC of the oregano leaves, most likely due to its high flavonoids content, was from 4 to 12 times higher than the ORAC _TAC of conventional forages, such as the fresh tops of trefoil

(ORACTAC = 330), lespedeza (ORACTAC = 530), and dried alfalfa hay (ORACTAC = 172 μιτιοΐ TE/g) (Ferreira, 2009). The ORACii _po values of OV were also higher than the ORACii _po usually obtained for conventional forage legumes and grasses such as lespedeza (ORACiipo = 9.28) and fescue (ORACi _ipo = 18.5 μιηοΐ TE/g). This high ORACi _ipo indicates the presence of EO components such as thymol and carvacrol.

Origanum vulgare spp. EO composition vastly differs between plants grown in different regions and climatic conditions, but generally the most biologically important compounds are thymol, carvacrol, p-cymene, and linalool (Vokou et al., 1993; Baser, 2002). Carvacrol was the main EO compound of the oregano used in this experiment. At 1.4% EO in OV DM, the concentration of carvacrol can be estimated at 12.7 g/kg OV DM. Thus, at 500 g/d (450 g DM) dietary supplementation, the cows received about 5.7 g/d carvacrol, which would correspond to about 57 mg L, assuming a 100-L rumen contents volume (and instantaneous consumption of the compound). We conducted an in vitro screening experiment to identify the antimethanogenic effect of the predominant compounds in oregano (procedures were similar to Tekippe et al., 2010). Of the 16 individual compounds screened, several (thymol, carvacrol, and geraniol) linearly decreased methane production within application rates of 400 to 4,000 mg/L final medium concentration (Hristov et al., unpublished). Several other compounds had less pronounced effect (linalool, for example) and some (p-cymene) had no effect on methane production. Carvacrol at 400 mg/L reduced methane production by 45% and completely inhibited it at 2,000 mg/L. These high concentrations cannot be practical for in vivo conditions and the effects observed in vitro cannot be reliably extrapolated to the live animal. Factors such as duration of feed (or feed supplement) consumption and ruminal turnover rate additionally make these comparisons difficult. Nevertheless, the observed level of methane inhibition in this study suggests that (1) carvacrol is not the only bioactive compound in OV having inhibitory effect on rumen methanogenesis, or (2) longer adaptation may be required for manifestation of the effect of carvacrol. Lambert et al. (2001), for example, combined thymol with carvacrol and observed higher antibacterial activity than either compound supplied separately. The effect of carvacrol, as a major compound of several EO, on rumen fermentation has been studied both in vitro and in vivo. Benchaar et al. (2007b) reported that oregano oil (200 mg/L), or carvacrol (400 mg/L) depressed in vitro DM or NDF degradability with no apparent effects on VFA production or molar ratios. Chaves et al. (2008) supplemented lamb diets with 0.2 g/kg DM carvacrol (which would

approximately correspond to the carvacrol concentration in the OV diet in this experiment), and found no effects on rumen fermentation, or lamb performance. Similar lack of effect on rumen fermentation was reported by the same group in continuous culture (Chaves et al., 2009). Methane production was not measured in these experiments, but an earlier attempt (Chiquette and Benchaar, 2005) reported a significant reduction in methane production by 225 to 250 mg/L carvacrol in vitro. Concentrations below 225 mg/L, however, had no effect on methane production, which is in support of the hypothesis that oregano EO, or carvacrol, were only partially responsible for the methane inhibition effect observed with oregano leaves in this experiment.

In spite of the significant reduction in ruminal methane production, there were no differences observed in archaeal diversity in this experiment. In an earlier study, there was no effect of coconut oil or lauric acid on methanogen-specific DGGE banding patterns despite a significantly lower methane production with coconut oil (Hristov et al., 2009). Long-term monensin supplementation reduced methane production by 7% (expressed as g/d), but when the diversity of methanogens was determined using DGGE targeting the methanogen 16S rRNA gene, no significant change was observed in the banding patterns (Hook et al., 2009). In the same study, the abundance of methanogens as determined by using real-time PCR was not affected by treatment. In the current study, there was no effect of OV on apparent NDF digestibility, or VFA concentration in the rumen, suggesting that there was no toxic effect of OV on fibrolytic bacteria, which are the main bacterial hydrogen producers in the rumen. It is possible that the inhibition of methane production by OV may have been due to a disruption in inter-species hydrogen transfer between fibrolytic bacteria and methanogens rather than a direct toxic effect on methanogens. It has to be also pointed out that the molecular techniques used in this study are not strictly quantitative (Amend et al., 2010) and we cannot be certain that treatments did not affect the size of the bacterial or archaeal populations.

The OV supplement had no effect on intake, or total tract digestibility of nutrients in the current experiment. We are not aware of other studies on the effect of OV on nutrient digestibility in cattle. Ropadiar (a commercial product based on oregano EO) supplemented to a sheep diet at 250 mg/d had no effect on total tract apparent digestibility of nutrients (Wang et al., 2009).

This is the first report characterizing the response of milk production, composition, and feed efficiency with OV supplementation. Other published research evaluated individual EO, including oregano, or EO active compounds. Spanghero et al. (2009), for example, fed a microencapsulated blend of EO that included oregano EO and found no effect on milk production or component yields. However, these authors reported a numerical trend for increased milk fat concentration. The effect on milk fat concentration and yield in the current study may be attributable to slight shifts in microbial fermentation in the rumen, evident in the numerical increase in acetate to propionate ratio in the OV treatment. The fat yield increase, paired with a numerical increase in milk production, resulted in a significant increase in the 3.5% FCM yield for the OV diet. As methane production constitutes about 2 to 12% gross energy loss in cattle (Johnson et al., 1994), the increased FCM yield may be partially attributed to increased metabolizable energy supply as a result of reduced methane production by OV.

The oregano fed to the cows in this experiment had no effect on the organoleptic characteristics of milk. Sensory differences are typically seen in milk from animals consuming feeds that contain plant terpenes, which are the major component of EO.

Thymol, for example, is a monoterpene. These compounds pass easily into milk (Viallon et al., 2000) causing off flavors. Thymol, however, was not found in EO from the OV used in this experiment.

CONCLUSIONS

Supplementation of dairy cow diet with 500 g/d of oregano leaves tended to increase milk fat percentage and increased fat yield, 3.5% FCM yield, and FCM feed efficiency. Due to a combination of insignificant, but unidirectional effects, the efficiency of transfer of feed N into milk protein was also increased by the oregano treatment.

Ruminal fermentation effects of the oregano were subtle, except ammonia concentration was increased, which resulted in increased plasma urea N concentration. We observed a sizable reduction in rumen methane production with the oregano supplementation during the 8 h sampling period; this effect, however, has to be interpreted with caution due to the large within and between animal variability in methane emission estimates. REFERENCES

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Table 1. Ingredient and chemical composition of the basal diet fed to cows during the trial.

Item Diet

Ingredient composition, % of DM

Corn Silage ¹ 42.4

Alfalfa Silage ² 13.1

Grass/Straw Hay 4.4

Canola Meal 7.5

Bakery by-product ³ 6.6

Ground Corn 7.4

Cottonseed Hulls 10.8

Sugar blend ⁴ 4.2

Min/Vit Mix ⁵ 3.2

Optigen ⁶ 0.4

Chemical composition (% of DM or as indicated)

DM, % 51.0

CP 15.6

ADF 20.5

aNDF 32.0

NFC ⁷ 43.7

Ether extract ⁷ 3.8

NE _L , Mcal/kg DM ⁸ 1.54

Ca ⁷ 0.93

P ⁷ 0.39

'Contained: 32.2% DM and (%, DM basis): 6.8 CP, 25.1 ADF, 40.0 aNDF, 48.4 NFC, 3.3 ether extract, 2.8 ash, 0.15 Ca, and 0.21 P.

Contained 47.3% DM and (%, DM basis): 20.4 CP, 30.6 ADF, 39.7 aNDF, 31.2 NFC, 2.8 ether extract, 9.7 ash, 1.22 Ca, and 0.30 P.

³ Bakery byproduct (Bakery Feeds, Honey Brook, PA) contained (%, DM basis): 9.0 CP, 8.0 ether extract, and 5.0 crude fiber.

⁴ Sugar blend (Westway Feed Products, Tomball, TX) contained (%, DM basis) 3.9 CP and 66.0 total sugar.

5Premix contained: 0.29% S, 7.183 mg/kg of Se, 4.7 mg/kg of Co, 11.76 mg/kg of I, 21.1 mg/kg of vitamin A, 438.6 μg kg of vitamin D, and 685.9 mg/kg of vitamin E.

⁶ Optigen is a non-protein nitrogen source (243% CP, DM basis) from Alltech, Inc.

(Nicholasville, Kentucky).

⁷ As analyzed by Cumberland Valley Analytical Services (Maugansville, MD).

8Estimated based on NRC (2001). Table 2. Chemical composition of Origanum vulgare L. and fescue hay used in the study

Item Fescue hay Origanum vulgare

L.

DM, % 89.3 89.9

Composition, % of DM

CP 5.8 10.9

ADF 47.0 22.2

aNDF 75.2 31.9

NFC 14.7 46.6

Ether Extract 2.0 1.4

Starch 2.3 8.5

Ash 4.2 9.2

Ca 0.3 2.1

P 0.1 0.1

As analyzed by Cumberland Valley Analytical Services (MaugansviUe, MD).

Table 3. Composition of essential oil extracted from Origanum vulgare L. leaves used the study (average of 3 replicated analyses)

Compound Concentration,

% of total

essential oil ¹

Carvacrol 90.8

γ-terpinene 2.34

δ-amorphene 1.50

β-caryopyllene 0.81

a-guainene 0.72

p-cymene 0.48

Borneol 0.48

τ-cadinol 0.32

trans Sabinene hydrate 0.27

Spathulenol 0.23

Carvacrol acetate 0.23

Caryophyllene oxide 0.18

γ-cadinene 0.11

δ-cadinene 0.12

Manool oxide 0.09

Unknown 1.59

Essential oil represented 1.4% oregano leaves DM.

2n = 3, SE = 4.1

Table 4. Ruminal fermentation characteristics of cows fed control or Origanum vulgare L - supplemented diets ( H, ammonia, and VFA, n = 60 ¹ ; protozoal counts and TFAA, n = 12; methane production, n = 50 ² )

Item Control OV ³ SEM P-value pH 6.0 6.1 0.09 0.47

Ammonia, mM 4.5 5.5 0.46 <0.001

TFAA, mM ⁴ 3.1 3.0 0.57 0.81

VFA, mM

Total 132.2 128.9 6.64 0.28

Acetate 80.8 79.7 2.25 0.20

Propionate 30.5 28.4 1.73 0.15

Butyrate 14.2 14.5 0.34 0.37

Isobutyrate 1.12 1.15 0.05 0.45

Valerate 3.24 2.77 0.32 0.14

Isovalerate 2.32 2.35 0.10 0.50

Acetate :propionate 2.72 2.86 0.29 0.10

Protozoa, x 10 ⁵ /ml 2.9 3.0

Log Protozoa 5.4 5.4 0.06 0.59

Methane production, g/h ⁵ 31.2 18.8 8.21 0.004

Represents number of observations used in the statistical analysis.

Measurements for one cow on the control treatment were lost during Period 1 of the trial. Another 4 individual time-point measurements were also lost and one observation was removed as an outlier based on an absolute studentized residual value > 3 (PROC REG of SAS); thus, n = 50.

3OV= 500 g d Origanum vulgare L.

4Total free amino acids.

⁵ Data collected within 8 h after feeding. Treatment x time interaction, P = 0.05 (see Fig. 2).

Table 5. Predominant (as % of total isolates) bacteria and archaea in whole ruminal contents of dairy cows fed control or Origanum vulgar e L.- supplemented diets (n = 12 )

Microorganism Control OV ³ SEM P-value

Bacteria

Eubacteri m 2.9 2.8 0.41 0.93

Lachnospiracea 7.3 8.5 1.82 0.32

Ruminococcus 3.1 3.0 0.85 0.76

Ruminococcus albus 0.4 0.3 0.14 0.87

Prevotella 2.7 2.7 0.38 0.93

Prevotella ruminicola 1.5 1.0 0.49 0.34

Butyrivibrio fibrisolvens 1.6 1.9 0.39 0.07

Succiniclasticum ruminis 1.6 2.5 0.48 0.20

Clostridiales 14.9 15.9 2.77 0.64

Clostridium 6.6 7.6 1.13 0.003

Bacteroidacea 26.8 20.3 5.71 0.03

Bacteroides 2.8 3.6 0.78 0.002 Archaea

Euryarchaeota 16.0 17.9 1.99 0.30

Methanobrevibacter 18.6 20.6 1.31 0.38

Methanobacteriaceae 61.5 58.9 2.94 0.48

The % represents the percentage of the total sequences analyzed within the sample. Represents number of observations used in the statistical analysis.

OV= 500 g/d Origanum vulgare L.

Table 6. Dry matter intake and production parameters of cows fed control or Origanum vulgare L. -supplemented diets (milk yield, n = 112 ¹ ; all other variables, n = 16)

Item Control OV ² SEM P-value

DMI, kg/d 26.6 26.0 0.83 0.24

Milk, kg/d 43.6 44.1 3.58 0.61

Feed Efficiency ³ 1.66 1.71 0.07 0.11

Milk fat, % 3.12 3.28 0.028 0.09

Fat yield, kg/d 1.36 1.44 0.11 0.04

Milk true protein, % 2.93 2.94 0.010 0.61

Protein yield, kg/d 1.29 1.30 0.14 0.87

Milk lactose, % 4.77 4.75 0.005 0.43

Lactose yield, kg d 2.07 2.08 0.19 0.85

MUN, mg/lOO mL 13.3 13.3 0.82 0.93

3.5% FCM, kg/d 40.7 42.2 0.84 0.03

Feed Efficiency 1.53 1.63 0.01 <0.001

Milk NE _L , Mcal/d 28.3 29.2 3.47 0.28

3 Milk ÷ DMI.

Table 7. Estimated ruminal microbial protein outflow from the rumen and N losses in dairy cows fed control or Origanum vulgar e L.- supplemented diets (n = 16 ¹ )

Item Control OV ² SEM P-value

Urinary purine derivatives excretion, mM/d

Allantoin 413 378 60.7 0.47

Uric acid 86 78 9.3 0.18

Total purine derivatives 498 456 68.6 0.39

Microbial N flow, g/d 333 302 49.3 0.39

Urinary and fecal N excretion

Urine N, g/day 240 230 20.0 0.06

Urine N, % of N intake 32.2 31.5 0.66 0.48

Urea N, g/d 663 590 8.31 0.05

Urea N, % of urine N 27.2 26.0 0.98 0.35

Fecal N, g/d 290 280 40.0 0.42

Fecal N, % of N intake 40.4 41.9 3.19 0.53

Total N excretion, g/d 530 510 40.0 0.42

4PUN= plasma urea nitrogen.

Example 3

Milk Balanced Reference Duo-Trio Test

Summary

Milk samples from dairy cows fed oregano according to the invention, were tested against a control milk using a balanced reference duo-trio test design. Of the 48 panelists tested, 31 correct responses were required to establish significance at a=0.05. Only 26 correct responses were received so the Oregano milk is not considered significantly different from the control.

Subjects

Panelists were 48 persons. Panelists were untrained and recruited from a panel pre- screened for discrimination ability.

Products

Products were the control milk and milk from cows receiving oregano supplement, which were served as 2 oz. portions in 3.5 oz. clear plastic cups. During testing, prepared sample sets were kept under refrigeration prior to being served.

Procedure

A balanced reference duo-trio test was used to determine if the oregano test milk was significantly different from the control milk. Panelists were presented with three cups: the control and oregano milk samples labeled with 3 digit blinding codes and a labeled reference, which was either the control or oregano sample. The presentation order was counterbalanced with reference being presented in the left position, and panelists were instructed to identify the coded sample that was the same as the reference. Water and saltine crackers were provided as palate cleansers. Evaluations took place in individual testing booths using Compusense ® five software.

Analysis

Data was analyzed using Compusense® five release 4.8 software.

Results Panelists were asked to identify which coded sample was the same as the presented reference under the duo-trio test design. Of the 48 total responses collected, 31 correct responses, or those in which panelists matched the correct coded sample to the reference, were required to establish significance at a= 0.05.

Results, presented in Table 8, show that the oregano milk sample was not found to be significantly different from the control, as it received only 26 correct responses (P > 0.05).

Table 8: Results of balanced reference duo-trio test for oregano milk sample vs. a control milk.

Significant results are marked with an asterisk (p<0 05).

Appendix

Duo-Trio Test- Number of correct answers necessar to establish level of si nificance.