WO2018100386A1

WO2018100386A1 - Methods and compositions for improving feed outcomes

Info

Publication number: WO2018100386A1
Application number: PCT/GB2017/053622
Authority: WO
Inventors: Radek Messias BRAGANCA; Charles James NEWBOLD; Eva Ramos MORALES; Robert James Nash
Original assignee: Bangor University; Aberystwyth University
Priority date: 2016-12-01
Filing date: 2017-11-30
Publication date: 2018-06-07
Also published as: GB201620438D0; GB2572113A; GB2572113B; GB201909383D0; GB2557247A

Abstract

The present invention relates to a method for improving outcomes in ruminant feeding, the method comprising administering a composition to the ruminant, the composition comprising a glycosidase inhibiting iminosugar; wherein the improved outcome is selected from one or more of: inhibiting protozoal activity in the rumen of a ruminant; and/or improving ruminant feed efficiency; and/or improving growth performance of a ruminant; and/or reducing greenhouse gas emissions. The invention also relates to a composition comprising a saponin and a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar.

Description

Methods and Compositions for Improving Feed Outcomes

Field of the Invention

The present invention relates to a method of improving outcomes in ruminant feeding. The improved outcome may be selected from inhibiting protozoal activity in a rumen of a ruminant; and/or improving feed efficiency of a ruminant; and/or improving growth performance of a ruminant; and/or reducing greenhouse gas emissions from a rumen of a ruminant. The present invention further relates to a composition comprising a saponin and a glycosidase inhibiting iminosugar.

Background to the Invention

Ruminants are animals which are able to acquire nutrients from plant-based food by fermenting the food in a four-compartment fore stomach prior to digestion, the fore stomach is made up of the rumen, reticulum, omasum and then the abomasum (true stomach). The plant matter consumed by ruminants is generally high in cellulose and the ruminants depend on microorganisms in their guts to assist in the digestion of this cellulose.

The microbial ecosystem of the rumen is characterised by the existence of a range of microorganisms, including bacteria, protozoa, fungi and archaea. Within this ecosystem, protozoa feed on bacteria causing a rapid turnover of protein within the rumen and decreasing the supply of bacterial protein to the ruminant host. In addition, protozoa in the rumen are involved in methane production since the protozoa live in symbiotic association with methanogenic archaea. Methanogenesis in the rumen is thought to represent a 2-12% loss of energy intake, and is estimated to contribute up to 20% of total anthropotic methane emissions.

Elimination of the ciliate protozoa has been shown to increase microbial protein supply by up to 30% and reduce methane production by up to 11 % (1). Therefore, the elimination of ciliate protozoa from the rumen would lead to an increased production efficiency and sustainability of ruminant meat and milk for food, whilst reducing greenhouse gas emissions from the supply chain. However, no effective defaunating agent has been developed or commercialised to date.

Saponins are secondary compounds found in over 500 plants belonging to various families. Saponins have previously been shown to be potent anti-protozoal agents. However, there is evidence that saponins are degraded by the bacterial population of the rumen over time (2, 3). Saponins consist of a polycyclic aglycone (or sapogenin) attached to a carbohydrate unit consisting of a monosaccharide or smaller oligosaccharide entity. The aglycone portion of the saponins is either a steroid or triterpene.

The anti-protozoal effect of saponins is related to their interaction with the sterol moiety present in the membrane of the protozoa (3). The transient effect of the saponins appears to be due to the cleavage of the glycosidic bond of the saponins by microbes present in the rumen. This cleavage of the aglycone from the saponin results in a sapogenin which is ineffective in inhibiting protozoa. This cleavage of the saponin in the rumen builds up over a period of days such that the anti-protozoal effect of the saponins is lost.

It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.

Summary of the Invention

The present invention relates to compositions and methods for inhibiting protozoal activity in the rumen of a ruminant, improving ruminant feed efficiency, improving growth performance of a ruminant and/or reducing greenhouse gas emissions. The invention is based in part on studies by the inventors in which they have shown that glycosidase inhibitors, in particular glycosidase inhibiting iminosugars, can be used to directly inhibit rumen protozoa to inhibit the degradation of saponins in the rumen and prolong the anti-protozoal effect of saponins.

In a first aspect of the present invention there is provided a method for improving outcomes in ruminant feeding. The method comprises administering to the ruminant a composition comprising a glycosidase inhibiting iminosugar. The improved outcome is selected from one or more of the following:

• inhibiting protozoal activity in the rumen of a ruminant; and/or

• improving ruminant feed efficiency; and/or

• improving growth performance of a ruminant; and/or

• reducing greenhouse gas emissions.

In a second aspect of the invention there is provided the use of a composition comprising a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar, for improving outcomes in ruminant feeding, wherein the improved outcome is selected from one or more of the following:

• inhibiting protozoal activity in a rumen of a ruminant; and/or • improving feed efficiency of a ruminant; and/or

• improving growth performance of a ruminant; and/or

• reducing greenhouse gas emissions from a rumen of a ruminant.

In embodiments of the first and second aspect of the invention, the composition further comprises a saponin.

In a third aspect of the invention there is provided a composition comprising a saponin and a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar.

The inventors of the present invention have surprisingly shown that glycosidase inhibitors, particularly glycosidase inhibiting iminosugars, can be used to inhibit protozoal activity in the rumen of ruminants and thereby improve the feed efficiency and productivity of said ruminants. Even more surprisingly, the present inventors have shown a synergistic effect observed between such glycosidase inhibiting iminosugars and saponins in inhibiting protozoal activity and improving feed efficiency and productivity of ruminants. Without wishing to be bound by theory, the present inventors believe that the glycosidase inhibiting iminosugars of the present invention increase the longevity of the anti-protozoal effect of saponins by protecting saponins from cleavage in the rumen.

As will be appreciated, the methods and uses of the present invention, as outlined above, are non-therapeutic.

Also provided is a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar, for use in a method for improving outcomes in ruminant feeding.

Further provided is a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar, and a saponin, for use in a method for improving outcomes in ruminant feeding.

The term "outcomes in ruminant feeding" is set out above in relation to the first and second aspects of the invention.

Assessment of feed efficiency

Assessment of the feed efficiency of a ruminant can be achieved by many methods which would be known to the skilled person working in this field of technology. In particular, feed efficiency according to the present invention may be assessed by determining a feed conversion ratio (FCR), gain output/feed output, feed costs versus productivity, residual feed intake (RFI) or the difference between actual intake and predicted intake, for example.

Feed conversion ratio is a measure of an animal's efficiency in converting feed mass into increases of the desired output, for example the final mass of the animal or the milk output. Animals with a low FCR are considered to be efficient users of feed.

Therefore, in embodiments of the present invention an improved feed efficiency means that the FCR is reduced by at least 1 % compared to the FCR when the ruminant does not have a glycosidase inhibiting iminosugar or a glycosidase inhibiting iminosugar and a saponin administered. Preferably the FCR is reduced by at least 2%, 2.5%, 3% or 3.5%. More preferably the FCR is reduced by at least 4%.

An improvement in feed efficiency of a ruminant may result in a ruminant being required to be fed less to achieve the desired output, for example the final mass of the animal or the milk output. This would thereby be more economical for farmers, for example. Therefore, in embodiments the improved outcome may include reduced feeding costs.

Assessment of growth performance

Assessment of the growth performance of a ruminant can be achieved by many methods which would be known to the skilled person working in this field of technology. In particular, growth performance according to the present invention may be assessed by reviewing weight gain of the ruminant, carcass yield (i.e. meat yield), milk yield and/or wool yield for example.

Therefore, the present invention also provides a method for improving meat yield, milk yield and/or wool yield of a ruminant comprising administering a glycosidase inhibiting iminosugar to the ruminant. In embodiments such a method further comprises administering a saponin to the ruminant.

Assessment of greenhouse gas emissions

Methods for assessing greenhouse gas emissions will be well known to those skilled in the art. In embodiments of the invention the greenhouse gas emissions are methane emissions. Methods for assessing methane emissions include using chambers or polytunnels to collect emissions from animals and measure the methane concentrations therein, using sulphur hexafluoride tracers or using open-path lasers, for example.

In embodiments of the present invention a reduction in methane emissions means that the methane emissions are reduced by at least 2% compared to the methane emissions when the ruminant does not have a glycosidase inhibiting iminosugar or a glycosidase inhibiting iminosugar and a saponin administered. Preferably, the methane emissions are reduced by at least 3%, 4%, or 5%. More preferably, the methane emissions are reduced by at least 6%. Most preferably, the methane emissions are reduced by at least 10%.

Assessment of protozoal inhibition

Methods for assessing protozoal inhibition will be well known to those skilled in the art. In particular, methods for assessing protozoal inhibition may include monitoring protozoal motility or protozoal numbers, or measuring protozoal activity based on the ability of protozoa to break down radioactively labelled bacteria, for example.

In embodiments of the present invention inhibition of protozoa may indicate that protozoal motility is reduced by 5% compared to the protozoal motility when the ruminant does not have a glycosidase inhibiting iminosugar or a glycosidase inhibiting iminosugar and a saponin administered. Preferably, the protozoal motility is reduced by at least 10%, 12%, 14%, 16%, 18% or 20%. More preferably, the protozoal motility is reduced by 25%, 30%, 35% or 40%.

Reducing Ammonia Excretion

In embodiments of the invention, the improved outcome (in addition or as an alternative to those mentioned above) may include reducing ammonia excretion. As protozoa in the rumen cause protein turnover by predating on bacteria, reduction of protozoa may increase the nitrogen utilisation of the ruminant, thereby reducing ammonia-N excretion.

Methods for assessing ammonia excretion will be well known to those skilled in the art. In embodiments of the present invention, reduction of ammonia excretion may indicate the ammonia excretion is reduced by 10% compared to ammonia excretion when the ruminant does not have a glycosidase inhibiting iminosugar or a glycosidase inhibiting iminosugar and a saponin administered. Preferably, the ammonia excretion is reduced by at least 15%, 20%, 25% or 30%. More preferably the ammonia excretion is reduced by at least 40%. Ruminants

The present invention relates to compositions and methods for improving outcomes in ruminant feeding, particularly, inhibiting protozoal activity in a rumen of a ruminant and/or improving feed efficiency of a ruminant and/or improving growth performance of a ruminant and/or reducing greenhouse gas emissions from a rumen of a ruminant.

A ruminant (or ruminant like) animal is a mammal and for the purposes of the present invention includes cattle, sheep, goats, giraffes, yaks, deer, antelope, camels, buffalo, elk, bison, moose, alpacas and llamas.

In embodiments of the present invention, the ruminant animal is selected from cattle, sheep or goats. Preferably, the ruminant animal is domestic cattle, which includes beef cattle and dairy cows for example.

Saponins

The saponins according to the present invention can be derived from a variety of sources. For example saponins for use in the present invention may be obtained from plants (i.e. may be naturally occurring) or may be artificially synthesised using chemical techniques well known to the person skilled in the art.

Saponins which may be utilised in the present invention may be obtained from plants, for example plants of the following families: Amaryllidaccae, Araliaceae, Asparagaceae, Lillaecae, Quillajaceae etc. In embodiments, the saponins of the present invention are derived from ivy (for example Hedera helix), Yucca, Quillaja, fenugreek, liquorice, tea, soybeans, peas, yams, sugar beets, alfalfa, asparagus, aloe, vanilla, zhimu, citrus fruits etc.

In preferred embodiments, the saponins of the present are obtained from ivy, Yucca or Quillaja. In embodiments, the saponins are obtained from ivy. The inventors of the present invention have shown that the combination of saponins and glycosidase inhibitors, particularly glycosidase inhibiting iminosugars, can be used to inhibit protozoal activity in the rumen and improve rumen efficiency. Although these effects have been demonstrated using saponins obtained from a variety of sources, saponins obtained from ivy have surprisingly been shown to be particularly effective. In embodiments in which the saponins are obtained from plants (for example the plants mentioned above), they may be extracted from the plants. In such embodiments, the composition may comprise a plant extract, for example an ivy extract, the plant extract comprising a saponin. As will be appreciated by the skilled person, a plant extract according to the present invention may comprise one or more saponins and, in addition, may comprise other plant secondary compounds found naturally in plants (for example essential oils, tannins etc.).

Methods for extracting saponins from plant materials will be well known to the skilled person working in this technical field. Suitably, the saponins are extracted by way of aqueous or solvent extraction.

In embodiments in which the saponins are in the form of plant extracts (for example an ivy extract), it is preferred if the plant extract has a saponin content to provide at least around 0.5-1 % of the animal's dry matter intake.

In embodiments, the plant extracts may be purified to further concentrate one or more saponins and/or remove other non-saponin compounds found in the plant extract. Suitable methods for purification of said plant extracts may include taking dried plant material, defatting using a non-polar solvent, extracting in an alcoholic solvent (for example 70% ethanol or methanol) and filtering and drying the extract. Further purification can be achieved by using chromatographic methods such as silica or cellulose resins. Direct extraction of a suitable saponin-containing plant dried material such as ivy fruits in aqueous alcohol also provides a saponin rich extract.

In embodiments, one or more saponins may be isolated from the plant extracts using techniques including chromatographic methods such as centrifugal thin layer chromatography, preparative thin layer chromatography, high performance liquid chromatography etc.

The saponin may be a derivative and/or a salt of a naturally occurring saponin. Suitable derivatives may be obtained by oxidation, reduction and/or alkylation of the naturally occurring saponin, for example.

The saponin may be a triterpene saponin. In embodiments, the saponin is selected from any one or more of alpha-Hederin (an ivy saponin), (25S)-5p-spirostan^-ol 3-0-p-D-glucopyranosyl-(1->2)-p-D-glucopyranoside (a Yucca saponin), glycyrrhizin (a liquorice saponin), glycosides of a pentacyclic triterpenoid that is olean- 12-ene substituted by hydroxy groups at positions 3 and 16, an oxo group at position 23 and a carboxy group at position 28 (the 3β, 16α stereoisomer) (Quillaja saponin) and salts and/or derivatives thereof.

The structures of the aforementioned saponin compounds are given below: alpha-Hederin:

(25S)^-spirostan^-ol 3-0^-D-glucopyranosyl-(1->2)^-D-glucopyranoside:

glycyrrhizin:

In embodiments, the saponin is selected from any one or more of alpha-hederin, araloside A, astragaloside, bacoside A, cucurbitacin, eleutheroside, ginsenoside, gymnemic acid, gypenoside, 20-hydroxyecdysone, tangshenoside I, tinosporoside. withanolide, quillaic acid glycosides, yuccaloeside, furostanols.

In embodiments of the present invention the saponin may comprise an aglycone selected from the following structures or salts and/or derivatives thereof:

Suitable derivatives may be obtained by oxidation, reduction and/or alkylation of the naturally occurring saponin, for example.

In embodiments of the present invention the saponin may comprise a carbohydrate unit selected from the following structures or salts and/or derivatives thereof:

Glycosidase Inhibitors and Glycosidase Inhibiting Iminosugars

The term "glycosidase inhibitor" and "glycosidase inhibiting iminosugar" as used in the present invention is used to mean a compound, more particularly an iminosugar, which can inhibit glycosidase enzymes which catalyst the hydrolysis of glycosidic bonds. Techniques for determining whether a compound acts as a glycosidase inhibitor will be well known to the skilled person, but may include, for example use of substrates such as p-nitrophenyl-glycosides, where the presence of an inhibitor will reduce the release of the coloured p-nitrophenol when an appropriate glycosidase is present.

Iminosugars, also known as iminosaccharides, are analogues of sugars where a nitrogen atom has replaced an oxygen atom in the ring structure. Iminosugars can be found naturally in various plants.

The glycosidase inhibiting iminosugars according to the present invention can be obtained from a variety of sources. For example glycosidase inhibiting iminosugars for use in the present invention may be obtained from plants (i.e. may be naturally occurring) or may be artificially synthesised using chemical techniques well known to the person skilled in the art. Glycosidase inhibiting iminosugars which may be utilised in the present invention may be obtained from plants, for example the following plants: potatoes, Eugenia jambolana, Lycium chinense, mulberry, Alexa species, Adenophora species, Lonchocarpus felipei, Baphia nitida, Commelina communis L, Angylocalyx pynaertii, Hyancinthoides non-scripta, Scilla campanulata, Stevia rebaudiana. In preferred embodiments of the invention, the glycosidase inhibiting iminosugars are obtained from potatoes, mulberry or Stevia rebaudiana. In particularly preferred embodiments, the glycosidase inhibiting sugars are obtained from Stevia rebaudiana. The inventors of the present invention have shown that glycosidase inhibiting iminosugars alone, or the combination of saponins and glycosidase inhibiting iminosugars can be used to inhibit protozoal activity in the rumen and improve rumen efficiency. Although these effects have been demonstrated using glycosidase inhibiting iminosugars obtained from a variety of sources, iminosugars obtained from Stevia rebaudiana have surprisingly been shown to be particularly effective.

In embodiments in which the glycosidase inhibiting iminosugars are obtained from plants (for example the plants mentioned above), they may be extracted from the plants. In such embodiments, the composition may comprise a plant extract, for example a Stevia extract, the plant extract comprising a glycosidase inhibiting iminosugar. As will be appreciated by the skilled person, a plant extract according to the present invention may comprise one or more glycosidase inhibiting iminosugars and, in addition, may comprise other plant secondary compounds found naturally in plants (for example essential oils, tannins etc.).

Methods for extracting glycosidase inhibiting iminosugars from plant materials will be well known to the skilled person working in this technical field. Suitably, the glycosidase inhibiting iminosugars are extracted by way of providing plant material, grinding or macerating, adding water or an aqueous solvent (such as 50% aqueous ethanol) and, after an appropriate time for extraction, filtering to obtain an extract containing iminosugars. The extract may then be dried by spray drying or vacuum drying for example.

In embodiments in which the composition (for example the composition for use in the method of the present invention) comprises a plant extract comprising a glycosidase inhibiting iminosugars (for example a Stevia rebaudiana extract), it is preferred if the plant extract has a glycosidase inhibiting iminosugar concentration of at least 1 mg/g, preferably at least 1.2, 1.4, 1.6 or 1.8 mg/g. Even more preferred is if the plant extract has a glycosidase inhibiting iminosugar content of at least 2 mg/g, 2.5 mg/g or 3 mg/g. In embodiments, the plant extracts may be purified to further concentrate one or more glycosidase inhibiting iminosugars and/or remove other non-glycosidase inhibiting iminosugar compounds found in the plant extract. Suitable methods for purification of said plant extracts may include use of a cation exchange resin or filtration to concentrate low molecular weight components, for example.

In embodiments, one or more glycosidase inhibiting iminosugars may be isolated from the plant extracts using techniques including chromatographic methods such as centrifugal thin layer chromatography, preparative thin layer chromatography, high performance liquid chromatography etc.

In preferred embodiments, the composition may comprise a Stevia rebaudiana extract.

Therefore, the present invention also relates to a method of improving outcomes in ruminant feeding. The method comprises administering a Stevia extract to the ruminant. In embodiments, the Stevia extract includes one or more glycosidase inhibiting iminosugars.

The definition of the term "improving outcomes in ruminant feeding" is set out above in relation to the first and second aspects of the invention.

As mentioned above, although the inventors have demonstrated that glycosidase inhibiting iminosugars obtained from a variety of sources can be used to inhibit protozoal activity in the rumen and improve rumen efficiency, saponins obtained from Stevia rebaudiana, for example Stevia rebaudiana extracts, have surprisingly been shown to be particularly effective and have an improved effect compared to glycosidase inhibiting iminosugars from other sources tested. In addition, Stevia obtained compounds and Stevia extracts have the added advantage of being accepted as a food additive, due to the FDA approved use of Sfew^'a as a natural sweetener and sugar substitute.

In embodiments of the present invention the glycosidase inhibiting iminosugar may comprise a compound of formula (I) or salts and/or derivatives thereof:

wherein R¹ is H, an optionally substituted Ci.io alkyl group or a CM_O alkyl carboxylate group; wherein R², R³, R⁴ and R⁵ are each independently selected from H, an optionally substituted CM_O alkyl group or an optionally substituted CM_O acyl group.

R¹ may be selected from a CM_O alkyl group, a CM₀ hydroxy substituted alkyl group, a CM_O alkyl carboxylate group.

R¹ may be a linear or branched optionally substituted CM_O alkyl group.

R¹ may be selected from ethyl, propyl, butyl, pentyl or hexyl groups.

R¹ may be a group of the formula -(CH₂)_nC0₂H , wherein n is an integer from 1 to 10.

Suitably, R¹ is -CH₂C0₂H.

Suitably, R¹ is H .

Suitably the compound of formula (I) has the following stereochemistry:

Suitably R², R³, R⁴ and R⁵ are all H. Suitably R¹ , R², R³, R⁴ and R⁵ are all H.

Suitably the glycosidase inhibiting iminosugar comprises a compound of formula (II) or salts and/or derivatives thereof:

Suitably the compound of formula (II) has the following stereochemistry:

The above compound is known as 2,5-dideoxy-2,5-imino-d-mannitol (DMDP).

In embodiments the glycosidase inhibiting iminosugar comprises a compound of formula (III) or salts and/or derivatives thereof:

Suitably the compound of formula (III) has the following stereochemistry:

The above compound is known as 2,5-dideoxy-2,5-imino-d-mannitol-N-acetic acid (DMDP- N-acetic acid).

In embodiments the glycosidase inhibiting iminosugar comprises a compound of formula (IV) or salts and/or derivatives thereof:

wherein R², R³, R⁴ are each independently selected from H, an optionally substituted CM₀ alkyl group or an optionally substituted CM_O acyl group.

Suitably the compound of formula (IV) has the following stereochemistry:

In embodiments the glycosidase inhibiting iminosugar comprises a compound of formula (V) or salts and/or derivatives thereof:

Suitably the compound of formula (V) has the following stereochemistry:

The above compound is known as Steviamine.

In embodiments the glycosidase inhibiting iminosugar comprises a compound of formula (VI) or salts and/or derivatives thereof:

wherein R is H, an optionally substituted CM_O alkyl group or a CM_O alkyl carboxylate group; wherein R⁷, R⁸, R⁹ and R¹⁰ are each independently selected from H , an optionally substituted Ci. ₁₀ alkyl group or an optionally substituted CM_O acyl group.

R^S may be selected from a CM_O alkyl group, a CM₀ hydroxy substituted alkyl group, a CM_O alkyl carboxylate group.

R^S may be a linear or branched optionally substituted CM_O alkyl group.

R^S may be selected from ethyl, propyl, butyl, pentyl or hexyl groups.

R^S may be a group of the formula -(CH₂)_nC0₂H , wherein n is an integer from 1 to 10.

Suitably, R⁶ is -CH₂C0₂H.

Suitably R⁶ is H.

Suitably the compound of formula (VI) has the following stereochemistry:

Suitably R⁷, R⁸, R⁹ and R¹⁰ are all H. Suitably R⁶, R⁸, R⁹ and R¹⁰ are all H.

Suitably the glycosidase inhibiting iminosugar comprises a compound of formula (VII) or salts and/or derivatives thereof:

Suitably the compound of formula (VII) has the following stereochemistry:

The above compound is known as 1-deoxynojirimycin (DNJ).

Suitable derivatives may be obtained by oxidation, reduction and/or alkylation of the compounds of formulas (l)-(VII), for example.

In embodiments of the present invention, the glycosidase inhibiting iminosugar comprises DMDP and/or DMDP-N-acetic acid.

In preferred embodiments, the glycosidase inhibiting iminosugar comprises DMDP. In such embodiments, the composition may comprise a plant extract, for example a Stevia extract, with a high DMDP content.

In embodiments, the concentration of DMDP in the plant extract is at least 1 mg/g, more preferably 1.2, 1.4, 1.6 or 1.8 mg/g, most preferably at least 2 mg/g, 2.5 mg/g or 3 mg/g.

Composition

The third aspect of the present invention relates to a composition comprising a saponin and a glycosidase inhibitor, preferably a glycosidase inhibiting iminosugar.

In embodiments of the invention the composition is a feed composition or feed additive, preferably a ruminant feed composition or ruminant feed additive. The invention also relates to a composition for improving outcomes in ruminant feeding. Outcomes in ruminant feeding are described in relation to the method and use of the present invention.

The composition of the present invention may be in dry or liquid form. For example, the composition may be in the form of a tablet, capsule, powder, solution or suspension.

In embodiments in which the composition is a feed composition or feed additive, the composition may be provided in admixture with animal food, or in a water supply (or other liquid feed) provided for an animal.

The saponins and/or glycosidase inhibitors (for example glycosidase inhibiting iminosugars) of the present invention may be incorporated into feed by methods known in the art of feed formulation and processing.

The composition or additive of the present invention may further comprise conventional ingredients present in feed compositions such as calcium carbonates, electrolytes such as ammonium chloride, proteins such as soya bean meal, wheat, starch, sunflower meal, corn, meat and bone meal, amino acids, animal fat, vitamins (such as vitamin A, vitamin D3, vitamin E, vitamin K, vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate) and minerals (such as trace minerals manganese, zinc, iron, copper, iodine, selenium, cobalt and/or macro minerals such as calcium, phosphorus and sodium).

In embodiments in which the composition is a ruminant feed composition or additive, the composition or additive may further comprise one or more ingredients commonly used in the ruminant diet, for example:

• Concentrate: largely comprises cereals (such as barley, maize, wheat, sorghum) and in addition, may further comprise protein-rich ingredients such as soybean, rapeseed, palm kernel, cotton seed and sunflower;

• Hay: comprises dry grass, legume and/or whole cereals. Grasses include timothy, ryegrasses and fescues, amongst others. Legumes include clover, Lucerne, alfalfa, peas, beans and vetches, amongst others. Whole cereals include barley, maize, oat and sorghum, amongst others; • Forage crops: including sugarcane, kales, rapes and cabbages;

• Root crops: including turnips, swedes, mangles, fodder beet and sugar beet (including sugar beet pulp and beet molasses);

• Tubers: including potatoes, cassava and sweet potatoes; and/or

• Silage.

In embodiments of the present invention the composition may comprise yet further components, such as antimicrobial agents, for example antibiotics.

The details set out above in relation to the composition of the third aspect of the present invention are equally applicable to the compositions of the method and use of the invention.

Dosage

The normal daily dosage of the glycosidase inhibiting iminosugar and/or saponin provided to the ruminant will depend upon the ruminant type and its condition.

In embodiments of the method of the present invention, the method may comprise administering the glycosidase inhibitor (preferably glycosidase inhibiting iminosugar) and/or saponin as a single daily or weekly dose, or alternatively, as a twice daily dose, given with the animal's feed, for example.

In embodiments of the invention, the composition may provide an overall dietary saponin intake of between 0.1 and 1 % of the animal's dry matter intake. In embodiments, the composition may provide an overall dietary glycosidase inhibiting iminosugar intake of between 0.2-2% of the animal's dry matter intake.

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

Moreover, any one or more of the above described preferred embodiments could be combined with one or more of the other preferred embodiments to suit a particular application. It should be understood that while the use of words such as "preferable", "preferably", "preferred" or "more preferred" in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as "a," "an," or "at least one," are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

Detailed Description of the Invention

The present invention will now be further described with reference to the following figures which show:

Figure 1 : Effect of ivy extract (2 g/l), either alone or combined with DNJ and DMDP (at 0.1 g/l), on protozoa motility over a 24 hour in vitro incubation;

Figure 2: The effect of the combination of ivy refined extract (1 mg/ml incubation) with DMDP (at 0.1 , 0.2, 0.4, 0.8 and 1 mg/ml incubation) on protozoa motility over time;

Figure 3: The effect of the combination of ivy refined extract (1 mg/ml incubation) with Stevia extract (0.2, 0.4, 0.8, 1 and 2 mg/ml incubation) on protozoa motility over time;

Figure 4: Protozoa motility over time in the presence of ivy refined extract (1 mg/ml) or three different Stevia extracts (Stein, SteChil and SteChi2), either alone or in combination;

Figure 5: Ammonia-N concentration (mM) in rumen content samples taken before and 2 hours after administration of ivy refined extract and Stevia extract, either alone or in combination, over a 14 day period;

Figure 6: The effect of ivy refined extract and Stevia extract, either alone or in combination, on ammonia concentration before (Oh) or 2 hours after (2h) the extract administration, over a 14 day period;

Figure 7: Protozoa numbers in rumen content samples taken before administration of ivy refined extract and Stevia extract, alone or in combination; Figure 8: Total volatile fatty acid concentration, acetic/propionic ratio and butyric acid concentration in rumen samples taken before (Oh) and 2 hours after (2h) administration of ivy refined extract and Stevia extract, alone or in combination;

Figure 9: Ammonia concentration (mM) in samples taken after 24 hours of in vitro incubations with ivy, Yucca or Quillaja (1 or 2 mg/ml) either alone or combined with Stevia (2 mg/ml);

Figure 10: Total VFA and butyric acid concentration (mM) and acetic/propionic ratio in samples taken after 24 hours of in vitro incubations with Ivy, Yucca or Quillaja (1 or 2 mg/ml) either alone or combined with Stevia (2 mg/ml);

Figure 11 : Protozoa motility over 24 hours in vitro incubations with ivy, Yucca or Quillaja (1 or 2 mg/ml) either alone or combined with Stevia (2 mg/ml);

Figure 12: Live weight gain for cattle fed on Yucca extract or Yucca extract and Stevia extract.

As discussed above, the anti-protozoal effect of saponins is transitory, as they become inactive when saponins are converted into the de-glycosylated sapogenin by rumen microorganisms. The present inventors undertook significant investigation to determine how protozoa of the rumen might be more effectively inhibited, how the anti-protozoal effect of saponins might be improved, and how outcomes in ruminant feeding more generally might be achieved.

Example 1 : In vitro effect of the combination of saponins with glycosidase inhibitors on protozoa and fermentation parameters over a 24 hour incubation

Initially, the inventors investigated the effect of ivy saponins, either alone or combined with two different glycosidase inhibiting iminosugars (DNJ and DMDP), on protozoa and fermentation parameters over a 24 hour incubation.

Strained rumen fluid from four cows (4 replicates) was diluted (1 :2) in artificial saliva solution (6). Aliquots (30 ml) of the diluted strained rumen fluid were added anaerobically to 120 ml Wheaton bottles containing 0.3 g of diet composed of ryegrass hay and barley (40:60), previously ground to pass through a 1 mm² mesh screen. Treatments consisted of control incubations (0.3 g of diet only), and incubations with Ivy (2 g/l) alone or combined with 0.1 g/l of DNJ or DMDP. Bottles were incubated at 39°C under C0₂ receiving a gentle mix before every sampling time. Samples at different time points (0, 4, 8 and 24 h) were collected for visual assessment of protozoa motility. Ciliate protozoa motility was assessed in 30 μΙ of sample against a common scale when examined at low magnification (x100) using light microscopy. This evaluation was conducted in less than 1 min/sample to avoid the cell damage originated by the oxygen and temperature exposure. A score between 0 (no protozoa evident) and 5 (all genera active) was given according to the scale described by Newbold (7). Fermentation pattern, in terms of pH and VFA was determined after 24 hours of the incubation. A subsample (4 ml) was diluted with 1 ml of deproteinising solution (200 ml/l orthophosphoric acid containing 20 mmol/l of 2-ethylbutyric acid as an internal standard) for the determination of VFA using gas chromatography (Stewart and Duncan, 1985). Another subsample (1 ml) was diluted with 0.250 ml. of trichloroacetate (25% (wt/vol) for analysis of ammonia using a colorimetric method (8).

Fermentation parameters were analysed statistically by randomised block ANOVA, with individual cows as a blocking term. Protozoal motility was analysed as a Repeated Measures Design, having treatment as main factor and incubation time as subject factor. When significant effects were detected across the different doses, means were compared by Fisher's protected LSD test.

The combination of ivy extract with the glycosidase inhibitors did not have a greater effect on fermentation parameters than when ivy extract was incubated on its own (Table 1). Regarding protozoal viability, glycosidase inhibitors DNJ and DMDP were more effective in reducing protozoa motility than when ivy was incubated on its own (Figure 1). Nevertheless, protozoal motility recovered after 24 hours for all treatments which may suggest that higher amount of glycosidase inhibitors should be added to the incubations to enable a more long term effect to be achieved.

Table 1 : Effect of Ivy (2 g/l) extract, either alone or combined with DNJ and DMDP (at 0.1 g/l), on pH, NH3-N and VFA profile in ruminal digesta after 24 hours of incubation

Control Ivy Ivy+DNJ Ivy+DMDP SED P

pH 6.37 6.31 6.32 6.34 0.031 0.299

Total VFA

(mmol/L) 80.8 75.3 76.9 79.4 2.76 0.245

VFA (%)

Acetate 67.9^C 62.3^a 61.6^a 63.2^b 0.382 <0.001

Propionate 16.0^a 22.2^C 22. 21.3^b 0.294 <0.001

Butyrate 11.9 11.5^a 11.7^ab 11.3^a 0.148 0.014

BCVFA 2.76 2.52 2.62 2.60 0.089 0.122

NH3-N

(mmol/L) 9.84^a 8 51^b 8.06 8 51^b 0.350 0.004

Example 2: In vitro studies of the effect of an ivy extract rich in saponins combined with increased concentrations of the glycosidase inhibitor DMDP on fermentation parameters and protozoal motility

The inventors mixed strained rumen fluid from four cows with artificial saliva solution (1 :2 dilution). Aliquots of 30 ml were incubated with 0.3 g of diet (barley:alfalfa hay, 60:40) under C0₂ and at 39°C for 24 hours. Treatments consisted of control incubations (diet only) and incubations with either a refined extract of ivy fruit alone (Hedera helix, 1 mg/ml) or in combination with DMDP at 0.1 , 0.2, 0.4, 0.6, 0.8 and 1 mg/ml. Samples were collected at 0, 4, 8 and 24 hours of the incubation to assess protozoa activity (Figure 1). Volatile fatty acid (VFA) concentrations were also determined (since ruminants obtain 70% of their energy from fatty acids) in addition to ammonia concentrations and pH (measured at 24 hours; Table 2). Fermentation parameters were analysed statistically by ANOVA considering treatments as main factor and cow as the block term. Fisher's protected least significance difference test was used to establish comparisons between treatments. Protozoal motility was analysed as a Repeated Measures Design, having treatment as main factor and incubation time as subject factor.

Neither ivy extract alone or in combination with DMDP caused an effect on total VFA or acetic acid concentrations (p>0.05; Table 2). However, incubations with ivy refined extract and DMDP at 0.6, 0.8 and 1 mg/ml resulted in an increase in propionic acid (p=0.023) and a subsequent decrease in the acetic/propionic ratio (p<0.001) as compared to ivy extract treatment alone. A reduction in butyric acid was also observed with increased concentrations of DMDP (p<0.001). Table 2: Effect of the combination of ivy refined extract ( 1 mg/ml incubation) with DMDP (at 0.1 , 0.2, 0.4, 0.6, 0.8 and 1 mg/ml incubation) on pH, VFA (mmol/l) and ammonia (mmol/l) after 24 hours of incubation

Different superscripts in a row indicate difference between means.

Additionally, the combination of ivy extract and DMDP at 0.6, 0.8 and 1 mg/ml caused a greater decrease in ammonia concentration and protozoa motility (Figure 2) over time than that of the ivy extract alone.

The cost of DMDP extraction is, at present, high. Therefore, the inventors undertook further testing on an extract of Stevia (Stevia rebaudiana Bertoni) leaves rich in iminosugars, in particular DMDP.

Example 3: Study of the effect of an ivy extract rich in saponins combined with increased concentrations of an extract of Stevia leaves rich in DMDP on fermentation parameters and protozoal motility

Incubations of 30 ml of inoculum (strained rumen fluid:buffer, 1 :2) with 0.3 g of diet (barley:hay, 60:40) and ivy refined extract (1 mg/ml) with or without Stevia extract (0.2, 0.4, 0.6, 0.8, 1 and 2 mg/ml incubation) were carried out as described above. Additionally Stevia extract (2 mg/ml incubation) was tested alone. Samples were collected at 0, 4, 8 and 24 hours of the incubation to assess protozoa activity (Figure 3). VFAs and ammonia concentrations were determined and pH measured at 24 hours (Table 3).

Fermentation parameters were analysed statistically by ANOVA considering treatments as main factor and cow as the block term. Fisher's unprotected least significance difference test was used to establish comparisons between treatments. Protozoal motility was analysed as a Repeated Measures Design, having treatment as main factor and incubation time as subject factor.

Stevia extract (2 mg/ml) incubated alone had an effect on fermentation parameters, increasing total VFA (p=0.024) and propionic acid concentrations (p<0.001) and decreasing butyric acid (p<0.001) in comparison with the control (see Table 3). The combination of ivy refined extract (1 mg/ml) with Stevia extract (2 mg/ml) altered fermentation towards propionate, reduced ammonia concentration, as expected when protozoa are inhibited/their number reduced, in a greater extent than that observed for ivy extract alone.

Table 3: Effect of the combination of different concentrations of Stevia extract with ivy refined extract on fermentation parameters and protozoa motility

Different superscripts within a row indicate difference between means.

As shown in Figure 3, Stevia extract alone (2 mg/ml) also decreased protozoal motility with a greater effect seen when a combination of Stevia extract (2 mg/ml) and ivy extract, indicating that saponins and glycosidase inhibiting iminosugars have a synergistic effect in inhibiting protozoa of the rumen.

Example 4: Study of the effect of an ivy extract rich in saponins combined with Stevia extracts differing in their content of glycosidase inhibitors on fermentation parameters and protozoa motility

Three Stevia extracts (Stein (India originating), SteChil and SteChi2 (China originating)) which differ in the concentration of glycosidase inhibiting iminosugars were tested. Stein has around five times more glycosidase inhibiting iminosugars than the SteChil and SteChi2 extracts. Further, the concentration of the major glycosidase inhibiting iminosugar DMDP in the Stein extract is around 2 mg/ml whereas the concentration of DMDP in the SteChil and SteChi2 extracts is around 0.8 mg/ml. SteChil and SteChi2 were richer in steviosides. Extracts were tested alone or combined with a saponin extract from ivy against protozoa over time. Fermentation parameters after 24 hours were also evaluated.

Incubations of 30 ml of inoculum (strained rumen fluid:buffer, 1 :2) with 0.3 g of a TMR diet (typically fed to lactating cows) and ivy refined extract (1 mg.ml) with or without 2 mg/ml of Stein, SteChil or SteChi2 were carried out as described above. Additionally, the three Stevia extracts (2 mg/ml) were incubated alone. Samples were collected at 0, 4, 8 and 24 hours of the incubation to assess protozoa activity (Figure 4). VFAs and ammonia concentrations were determined and pH measured at 24 hours (Tables 4 and 5).

When the different Stevia extracts were compared, Stein was found to be more effective in reducing protozoa motility than SteChil and SteChi2 (Table 4 and Figure 4). This is likely due to the high content of glycosidase inhibiting iminosugars in this extract.

Table 4: Effect of three different Stevia extracts (Stein, SteChil and SteChi2) on fermentation parameters at 24 h and protozoa motility over time.

Different superscripts within a row indicate difference between means.

The combination of ivy with Stein extract was more effective against protozoa than when Ivy was incubated alone. However, neither SteChil nor SteChi2 extracts appeared to improve the effectiveness of ivy saponins in inhibiting protozoal motility (Table 5 and Figure 4). Table 5. Effect of ivy extract, either alone or combined with each Stevia extract (Stein, SteChM and SteChi2) on fermentation parameters at 24 h and protozoa motility over time.

Different superscripts within a row indicate difference between means.

These studies indicate that the presence of glycosidase inhibiting iminosugars in Stevia extract inhibit protozoal motility and, in addition, increase the anti-protozoal effect of saponins.

Example 5: Study of the long term effect of ivy refined extract and Stevia extract, either on their own or combined, on protozoa numbers, fermentation parameters, digestibility and methane production

The inventors next assessed the effect of ivy and Stevia extracts, either on their own or in combination, on protozoa numbers, bacterial diversity and rumen fermentation in cannulated sheep. Nutrient digestibility and methane production were also studied.

The experimental design followed a replicated 4x4 Latin square design, with two replications (4 treatments, 4 periods, 8 experimental animals and two squares). Each period lasted for 21 days followed by a wash out period of at least two weeks to avoid the possible carry over effect of the treatments.

Sheep were fed a diet consisted of 1.2 kg of ryegrass hay and 250 g of sugar beet. The experimental diet was either given alone or supplemented with ivy refined extract, Stevia Stein extract or a combination of both, resulting in the following four treatments:

1. Control (experimental diet only);

2. Ivy (basal diet with 10 g/animal/day of Ivy extract);

3. Stevia (basal diet with 20 g/animal/day of Stevia extract); and 4. Ivy+ Stevia (basal diet plus 10 g/animal g/animal/day of Ivy extract and 20 g/animal/day of Stevia extract).

During each experimental period samples of rumen content were taken every day, before and two hours after feeding. Samples were analysed for VFA and ammonia-N and, in addition, protozoa number were counted. At the end of the experimental periods, methane production, nutrient digestibility and nitrogen utilization were also evaluated.

Statistical analyses of data were performed using the GLM procedure of Genstat 15th Edition, in a replicated Latin square design with two replicates (rows of different squares are independent but the columns are shared).

Effect of ivy refined extract and Stevia extract, either alone or in combination, on fermentation parameters and protozoa numbers

Ammonia concentration in rumen fluid samples taken before and 2 hours after feeding is shown in Figures 5 (concentration over time) and 6 (results of statistical analysis). There were no statistical differences in ammonia concentration between treatments (p=0.085) of samples taken before feeding (Oh), although a numerical decrease was observed for ivy and ivy+Sfew^'a treatments. Stevia extract caused an increase in ammonia concentration (P<0.001) in rumen samples obtained 2 hours after feeding, as compared with the control and the two other treatments. This is likely due to the Stevia extract being rich in nitrogen (1.6%). However, when Stevia extract was combined with ivy extract, ammonia concentration in samples either collected before or 2 hours after feeding, was reduced. Ammonia concentration in samples taken 2 hours after feeding was also numerically lower with the ivy treatment.

Regarding protozoa numbers in samples taken before feeding, although day to day variations in numbers were observed, values for animals corresponding to the control group were stable over the 14 days (Figure 7). Within the first half of the experimental period, there was a slight drop in protozoa numbers as a consequence of the administration of Ivy extract. In the presence of Stevia or Ivy+Sfew^'a protozoa numbers decreased between days 2 and 6 remaining lower that those corresponding to the control (Figure 7).

Total VFA and butyric acid concentrations as well as the acetic/propionic ratio over 14 days, in samples taken before and 2 hours after feeding are shown in Figure 7. None of the treatments had an effect on total VFA or butyric acid concentrations (p>0.05). However, a significant decrease (p<0.001) in acetic/propionic ratio was observed in rumen samples (Oh) of sheep receiving Stevia and ivy+Sfew^'a treatments in comparison with the control. The proportion of energy in fermented glucose retained as acetate, propionate and butyrate is 0.62, 1.09 and 0.78 respectively. Therefore, propionate formation conserves more of the energy present in fermented glucose in a useful form for the animal. In addition, propionate is the only glucogenic volatile fatty acid, accounting for 65-80% of the net glucose supply in lactating dairy cows. Therefore a decreased acetic/propionic ratio is indicative of increased availability of energy for the ruminants.

Effect of Ivy refined extract and Stevia extract, either alone or in combination, on methane production and nutrient digestibility

Methane production of sheep receiving ivy, Stevia or ivy+Sfew^'a extracts after 21 days is shown in Table 6. Although no significant differences (p>0.05) in methane production between treatments were observed, ivy+Sfew^'a treatment caused a decrease of 6 % as compared with the control.

Table 6. Effect of ivy, Stevia or ivy+Stevia on methane production

The effect of ivy and Stevia, either alone or in combination, on apparent nutrient digestibility and nitrogen utilization is shown in Tables 7 and 8. Our results showed that the supplementation of the diet with either ivy, Sfew^'a or a combination of both did not have an effect on nutrient digestibility, in comparison with the control (p>0.005) (Table 7). These results are in agreement with studies which investigated the use of saponins from other sources at different concentrations (4, 5). Table 7. Effect of Ivy, Stevia or Ivy+Stevia on apparent digestibility of nutrients

Regarding nitrogen utilization, although no significant differences between treatments in nitrogen intake or excretion were observed (p>0.05), faecal nitrogen was numerically lower (8% reduction) with the ivy+Stew^'a treatment in comparison with the control and the other two treatments (Table 8). Accordingly, digestible nitrogen as well as nitrogen retention was higher in animals receiving the ivy+ Stevia treatment.

Table 8. Effect of Ivy, Stevia or ivy+Siew^'a on metabolic weight and N balance

These examples show that Stevia extract, particularly extract which is rich in glycosidase inhibiting iminosugars such as DMDP, has an anti-protozoal effect. Furthermore, the combination of ivy extract and Stevia extract results in a shift of fermentation towards an increased production of propionate and a decrease in ammonia concentration (9%) and methane production (6%). These changes are likely due to the lower numbers of protozoa in the rumen. Although there appeared to be no change in nutrient digestibility resulting from treatment with ivy extract or Stevia extract (either alone or in combination), nitrogen retention was greater in sheep which had received treatment with saponins and glycosidase inhibiting iminosugars (in the form of ivy extract and Stevia extract). The combination of saponins and glycosidase inhibiting iminosugars, or glycosidase inhibiting iminosugars alone, could therefore be used in ruminant diets to reduce protozoa numbers, increase nitrogen utilisation and enhance animal production.

Example 6: Effect of the combination of three plant extracts rich in saponins (ivy, Yucca and Quillaja) with Stevia extract on protozoa motility and fermentation parameters over a 24 hour in vitro incubation

As discussed above, the combination of ivy saponins (1 mg/ml) with a Stevia extract (2 mg/ml) rich in the natural glycosidase inhibiting iminosugar DMDP, resulted in a greater decrease in acetic/propionic ratio and ammonia concentration at 24 hours than that caused by an ivy extract alone. In addition, at 8 and 24 hours of the incubation, protozoal motility decreased when the ivy and Stevia extracts were combined in comparison with the ivy extract alone.

The inventors then undertook further investigation to assess the effect of alternative saponin sources, Yucca and Quillaja, either alone or given in combination with a Stevia extract rich in DMDP, on protozoa motility and rumen fermentation parameters after 24 hours of incubation.

Rumen fluid was collected from 4 cannulated cows (n=4) strained through a double layer of muslin and diluted 1/3:2/3 with incubation solution (6). Thirty millilitres of diluted rumen fluid were added under C0₂ to wheaton bottles containing the experimental diet (TMR for cows-60:40, ryegrass silage oncentrate) and either ivy, Yucca and Quillaja (1 or 2 mg/ml) alone or combined with Stevia extract (2 mg/ml). Bottles were incubated at 39°C. Samples at different time points (0, 4, 8 and 24 hours) were collected for visual assessment of protozoa motility. Ciliate protozoa motility was assessed in 30 μΙ of sample against a common scale when examined at low magnification (x100) using light microscopy. This evaluation was conducted very quickly (less than 1 min/sample) to avoid the cell damage originated by the oxygen and temperature exposure. A score between 0 (no protozoa evident) and 5 (all genera active) was given according to the scale described by Newbold (7).

At the end of the experiment (24 hours), samples were collected to measure pH and analyse volatile fatty acids (VFAs) (1 ml 20% orthophosphosporic acid containing 20 mM 2 ethyl butyric acid + 4 ml of sample) by gas chromatography (9) and ammonia analysis (1 ml sample + 0.25 μΙ TCA) by phenol hypochlorite method (8). Fermentation parameters were analysed by ANOVA considering treatments as main factor and cow as the block term. Fisher's protected least significance difference test were used to establish comparisons between treatments. Findings with p<0.05 were considered as statistically significant. Data collected for protozoa motility were analysed by a repeated-measures ANOVA.

The inventors tested Yucca schidigera (rich in sterol saponins) and Quillaja saponaria (contains triterpene saponins), commonly used in ruminant nutrition, in 24 hour in vitro incubations (batch cultures). Ivy refined extract either alone or with Stevia, was also included in the incubation run. A total mixed ration (TMR: 60:40, ryegrass silage: concentrate) for cows was used as substrate of the fermentation. This diet is very different from the one that we have used in previous in vitro experiments (hay and barley: 40:60) and therefore the effect of the saponins on fermentation and protozoa may vary as it has been shown that the effect is diet dependent (10, 11).

Among the three sources of saponins tested, the greatest effect on fermentation parameters and protozoa activity was observed in the presence of ivy refined extract. A decrease in ammonia-N and butyric acid concentrations and acetic/propionic ratio was observed in the presence of ivy refined extract added at 1 mg/ml and Stevia extract added at 2 mg/ml, which is in agreement with results of our previous experiments (Figures 9 and 10). However, these reductions were more pronounced when ivy and Stevia were incubated with hay:barley (40:60) instead of TMR.

Yucca and Quillaja added at 2 mg/ml caused a reduction in ammonia concentration and acetic/propionic ratio in comparison to the control, being these effects greater with the Yucca extract (Figures 9 and 10). Protozoa motility was slightly affected by the addition of Yucca and Quillaja at 2 mg/ml and no effect was observed on butyric acid concentration (Figures 10 and 11). When Yucca and Quillaja extracts were added in combination with Stevia extract, the reduction in ammonia, acetic/propionic acid and protozoa motility was greater than when they were added alone (Figures 9, 10 and 11).

Based on these results the inventors concluded that Yucca and Quillaja saponins could be used as an alternative to ivy based saponins in combination with glycosidase inhibiting iminosugars to improve ruminant feed efficiency and inhibit protozoal activity in the rumen. Example 7: Effect of the combination of ivy saponins and a Stevia extract rich in glycosidase inhibiting iminosugars on growth of beef cattle fed on TMR

The inventors then went on to perform an in vivo beef cattle trial, in which they utilised saponins in the form of Yucca extract either alone or in combination with a Stevia extract (Stein) high in glycosidase inhibiting iminosugars.

For the majority of the trial, animals on the saponin and Stevia treatment had higher DM intakes compared to the control and saponin treatments (data not shown). This resulted in an increased live weight gain for the animals treated with Yucca saponins and Stevia extract compared to the control animals or those treated with Yucca saponins only (Figure 12), indicating that the combination of saponins and glycosidase inhibiting iminosugars can be used to improve ruminant productivity.

It will be appreciated that numerous modifications to the above described compositions, method and uses may be made without departing from the scope of the invention as defined in the appended claims. For example, although particular saponins (derived from ivy, Yucca and Quillaja) and glycosidase inhibiting iminosugars (from Stevia) have been exemplified, it will be appreciated that saponins and glycosidase inhibiting iminosugars from other sources could be utilised in the present invention.

References

1. Newbold CJ, de la Fuente G, Belanche A, Ramos-Morales E and McEwan NR (2015). The Role of Ciliate Protozoa in the Rumen. Front Microbiol. 26;6: 1313. doi: 10.3389/fmicb.2015.01313.

2. Newbold, C.J., El Hassan, S.M., Wang, J., Ortega, M.E. and Wallace, R.J. (1997). Influence of foliage from African multipurpose trees on activity of rumen protozoa and bacteria. The British Journal of Nutrition 78, 237-249.

3. Patra K.A.,and Saxena, J. (2009). Dietary phytochemicals as rumen modifiers: a review of the effects on microbial population. Antonie van Leeuwenhoek 96, 363-375.

4. Aazami MH, Tahmasbi AM, Ghaffari MH, Naserian AA.Valizadeh R and Ghaffari AH. (2013). Effects of Saponins on Rumen Fermentation, Nutrients Digestibility, Performance, and Plasma Metabolites in Sheep and Goat Kids. Annual Review & Research in Biology 3(4): 596-607.

5. Wina E, Muetzel S and Becker K. (2006). Effects of Daily and Interval Feeding of Sapindus rarak Saponins on Protozoa, Rumen Fermentation Parameters and Digestibility in Sheep. Asian- Aust. J. Anim. Sci. Vol. 19, No. 11 : 1580 - 1587.

6. Menke, K. H., and Steingass, H. (1988). Estimation of the energetic feed value obtained from chemical analysis and gas production using rumen fluid. Anim. Res. Dev. 28, 7-55.

7. Newbold, C. J. (2010). "Assessing antiprotozoal agents" in: In vitro Screening of plant resources for extra-nutritional attributes in ruminants, ed P. E. Vercoe, H. P. S. Makkar, and C. Schilink, 47-53.

8. Weatherburn MW, Phenol-hypochlorite reaction for determination of ammonium. Anal Chem 39: 971-974 (1967).

9. Stewart, C.S., and Duncan, S.H. (1985). The effect of avoparcin on cellulolytic bacteria of the ovine rumen. J. Gen. Microbiol., 131 , 427-435. 10. Goel, G., Makkar, H. P. S., and Becker, K. (2008). Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin-rich fractions from different plant materials. J. Appl. Microbiol. 105, 770-777.

1 1. Szczechowiak, J., Szumacher-Strabel, M., Stochmal, A., Nadolna, M., Pers-Kamczyc, E., Nowak, A., Kowalczyk, M., Cieslak, A. (2013). Effect of Saponaria officinalis L. or Panax ginseng C.A Meyer triterpenoid saponins on ruminal fermentation in vitro. Ann. Anim. Sci., 13, 815-827

Claims

A method for improving outcomes in ruminant feeding, the method comprising administering a composition to the ruminant, the composition comprising a glycosidase inhibiting iminosugar;

wherein the improved outcome is selected from one or more of:

inhibiting protozoal activity in the rumen of a ruminant; and/or improving ruminant feed efficiency; and/or

improving growth performance of a ruminant; and/or

reducing greenhouse gas emissions.

Use of a composition comprising a glycosidase inhibiting iminosugar for improving outcomes in ruminant feeding, wherein the improved outcome is selected from one or more of the following:

inhibiting protozoal activity in a rumen of a ruminant; and/or

improving feed efficiency of a ruminant; and/or

improving growth performance of a ruminant; and/or

reducing greenhouse gas emissions from a rumen of a ruminant.

The method or use of claim 1 or 2 wherein the composition further comprises a saponin.

A composition comprising a saponin and a glycosidase inhibiting iminosugar.

The method, use or composition according to any preceding claim wherein the glycosidase inhibiting iminosugar is obtained from a plant.

The method, use or composition according to any preceding claim wherein the composition comprises a Sfew^'a rebaudiana extract.

The method, use or composition according to any preceding claim wherein the glycosidase inhibiting iminosugar comprises a compound of formula (I) or salts and/or derivatives thereof:

wherein R¹ is H or an optionally substituted C1.10 alkyl group or a Ci .i o alkyl carboxylate group; wherein R², R³, R⁴ and R⁵ are each independently selected from H, an optionally substituted Ci .i o alkyl group or an optionally substituted C1.10 acyl group.

8. The method, use or composition according to claim 7 wherein the glycosidase inhibiting iminosugar comprises 2,5-dideoxy-2,5-imino-d-mannitol (DMDP).

9. The method, use or composition according to claim 7 wherein the glycosidase inhibiting iminosugar comprises 2,5-dideoxy-2,5-imino-d-mannitol-N-acetic acid (DMDP-N-acetic acid).

10. The method, use or composition according to any one of claims 1-6 wherein the glycosidase inhibiting iminosugar comprises a compound of formula (IV) or salts and/or derivatives thereof:

wherein R², R³, R⁴ are each independently selected from H, an optionally substituted CM₀ alkyl group or an optionally substituted CM₀ acyl group.

1 1. The method, use or composition according to claim 10 wherein the glycosidase inhibiting iminosugar comprises Steviamine.

12. The method, use or composition according to any one of claims 1-5 wherein the glycosidase inhibiting iminosugar comprises a compound of formula (VI) or salts and/or derivatives thereof:

wherein R is H, an optionally substituted Ci.₁₀ alkyl group or a Ci.i₀ alkyl carboxylate group; wherein R⁷, R⁸, R⁹ and R¹⁰ are each independently selected from H, an optionally substituted C₁-₁₀ alkyl group or an optionally substituted C₁-₁₀ acyl group.

13. The method, use or composition according to claim 12 wherein the glycosidase inhibiting iminosugar comprises 1-deoxynojirimycin (DNJ).

14. The method, use or composition according to any one of claims 3 or 4 or 5-13 when dependent on claims 3 or 4 wherein the saponin is obtained from ivy, Yucca or Quillaja.

15. The method, use or composition according to claim 14 wherein the composition comprises an ivy extract.

16. The method, use or composition according to any one of claims 3 or 4 or 5-15 when dependent on claims 3 or 4 wherein the saponin comprises a compound of the following structure or salts and/or derivatives thereof: