This invention relates to animal feedstuff additives, particularly ruminant feedstuff additives, their manufacture, feedstuffs containing them and methods of animal husbandry, particularly of ruminants, using the compositions. It is known to include choline as a dietary supplement in feed for domesticated animals such as pigs and poultry. Such choline supplements are usually of choline chloride supported on an inert carrier such as com husks or silica to give free flowing granules. Canadian Patent No 0943460 describes free flowing granules containing choline chloride made by spray drying an aqueous solution containing choline chloride and a fatty acid soap, e.g. the sodium salt of a mixture of C ₁₆ and C ₁₈ fatty acids.

B K Sharma et al (Journal of Dairy Science, Vol 71 , No 9, 1988) describe that direct addition of choline into the abomasum of a ruminant such as a cow, produced higher yields of milk fat compared with the direct addition of methionine. However, in ruminants choline is destroyed by the microorganisms in the rumen and choline in feed is destroyed before it can be absorbed in the intestine. Thus, adding choline to feed is not an effective way of supplementing the diet of ruminants. British Patent No 2005537 A describes rumen protected pellets in which a core material is enclosed in a polymeric coating. The core material includes an active agent, e.g. methionine, and a basic material to raise the core material pH above 5 pH. An inert inorganic substance such as clay, may be included to adjust the density. The protective polymeric coating includes a polymer and when the active agent is acidic and water soluble may also include up to 50% by weight of a hydrophobic substance. British Patent No 1387038 describes pelleted materials containing an active agent such as glucose, with a protective coating of a mixture of saturated and unsaturated fatty acids, or their salts.

We have found that direct mixing of choline for example as choline chloride, with a fatty acid does not prevent destruction of the choline in the rumen when fed as part of a dietary supplement and that using alkali or alkaline earth metal soaps of the fatty acid(s) does not give a feedstuff additive having adequate resistance to degradation in the rumen.

Surprisingly, we have now found that a feedstuff additive including a choline compound may be adequately protected from destruction in the rumen by supporting the choline compound on a suitable carrier and then coating the carrier with a protective matrix including at least one fatty acid or fatty acid soap.

Accordingly, the present invention provides a ruminant feedstuff additive, which comprises a post-ruminally effective choline compound releasably supported on an acceptable carrier, coated by or dispersed within a protective matrix having a melting point of at least 40"C, the matrix being made up of one or more water insoluble fatty acid(s) or fatty acid salt(s), the protective matrix comprising at least 35% by weight of the additive.

The invention also provides a ruminant feedstuff which includes a feedstuff additive of the invention typically in combination with other dietary materials. The invention further includes a

method of animal husbandry which comprises feeding to a ruminant a feedstuff additive or a ruminant feedstuff of the invention.

The invention includes a method of preparing a feedstuff additive of the invention comprising releasably supporting the choline compound on the acceptable carrier and incorporating the releasably supported choline compound within the protective matrix.

The acceptable carrier is typically an absorbent relatively inert material having pores that can absorb liquid. Suitable materials include inorganic materials, such as hydrophilic silica, diatomaceous earth, bentonite and other clays and lime (CaCO- _j ), and organic materials, including cellulosic materials, particularly cellulosic residues of processed vegetable matter such as com (maize) cobs, coconut husk, bagasse, sugar beet fibre and dried grape skins. Paniculate precipitated hydrophilic silica and/or ground com cobs are particularly suitable carriers. Suitable cellulosic carriers are typically residues from the treatment of agricultural products and commercially are often dried before sale. With such dried cellulosic carriers, we have found it advantageous to wet the carrier to improve its hydrophilicity prior to supporting the choline compound on it. The carrier will typically have a particle size of from 30 to 500μm, more usually from 50 to 250μm.

The post-ruminally effective choline compound used in the additive composition can be any that provides a physiologically acceptable source of choline to a ruminant. Suitable compounds include choline chloride, choline dihydrogeπ citrate, tricholine citrate and choline bitartrate. Choline chloride is readily available and has a high specific choline content and is a particularly suitable choline compound for use in this invention. Solid choline chloride is a deliquescent crystalline material and is very soluble in water (about 80 g.100 ml ^" at 20°C). Solutions of choline chloride in water are very nearly neutral. A near saturated aqueous solution of choline chloride e.g. about 75% by weight, is a convenient source of choline chloride in making the additive composition of the invention. Aqueous choline chloride solution at a concentration of about 75% by weight and having a pH of from 6 to 8 is the normal form of choline chloride sold and transported in commerce. Typically the choline compound will be used in an amount of from 40 to 70%, more usually from 40 to 60%, and very suitably about 50% by weight of the combined weight of carrier and choline compound. The protective matrix has a melting point of at least 40 ^o C, preferably from 40 to 65 ^β C, for example from about 50°C to about 60 ^β C, and is formed from one or more fatty acids and/or or fatty acid salts. A suitable matrix is one which is a solid under ambient conditions to give an additive with an easily handled form and is resistant to the environment within the rumen. It is important that the matrix is not only resistant to attack by the microorganisms and chemicals in the rumen but is also able to retain its physical integrity. The physical integrity of the matrix depends on the temperature and typical matrix materials are solid under ambient conditions, soften as the temperature increases and eventually melt. The matrix material used in the invention has a melting temperature of at least 40°C as the typical rumen temperature is about 40°C. Materials with lower melting temperatures do not provide effective protection to the supported choline in the rumen. Suitable fatty acids for use in

the protective matrix include palmitic acid, stearic acid, mixtures containing them e.g. those in some naturally occurring oils, and their salts. The protective matrix may also be formed from mixtures of fatty acids which include unsaturated acids such as oleic acid, e.g. as found in palm oil, provided that the melting temperature of the matrix is at least 40°C. Saturated fatty acids generally have higher melting points than the corresponding unsaturated acids. Thus, an initially unsatisfactory unsaturated fatty acid or mixture may be hydrogenated to give a fatty acid or mixture having a suitably higher melting temperature. The protective matrix can be wholly of the fatty acid, a mixture of fatty acid and fatty acid salt or of fatty acid salt. Where the matrix includes fatty acid salts, either the salts will themselves be insoluble or the proportion of salt will not be such that its solubility causes failure of protection by the fatty acid(s). As is described below, small proportions of soluble salts may make the fatty acid matrix less susceptible to disruption in the rumen by plasticising the matrix.

The amount of protective matrix in the additive composition is at least 35% but usually will not be more than about 80% by weight of the additive composition. More usually, the amount is from 35 to 60%. Amounts less than about 35% do not give satisfactory protection in the rumen to the choline compound. Amounts above about 80% limit the amount of choline compound that can be included in the additive. We have found that, where the protective matrix is from 35 to 50% of the weight of the feedstuff additive typically up to about 50% of the choline compound is available for post-ruminal assimilation and where the protective matrix is from 50 to 60% of the weight of the feedstuff additive up to about 90% of the choline compound is available for post-ruminal digestion. The additive composition contains the choline compound releasably supported on an acceptable carrier. In order to achieve adequate protection of the choline from degradation in the rumen, the supported choline compound will usually be prepared before coating by or incorporation into the matrix. Incorporation within or coating by the protective matrix of the supported choline compound may be carried out by mixing the supported choline compound with heated molten protective matrix material and thereafter cooling and solidifying the mixture to form the desired additive composition. One effective way of doing this is to continue to heat the supported choline compound and molten protective matrix material during mixing so that free water present is evaporated from the mixture. Alternatively, the supported choline compound and protective matrix may be coextruded under conditions such that the protective matrix softens sufficiently to allow satisfactory incorporation of the supported choline compound within the matrix material. Other techniques that can be used include pellet milling and granulation. If the choline compound is not supported on the carrier prior to incorporation into the matrix material the protection of the choline compound may end up at least in part not being supported on the carrier and this will usually mean that it is not adequately protected from degradation in the rumen.

We have obtained good results if the choline compound is supported on the carrier initially as an aqueous solution e.g. by mixing a solution of the choline compound with the carrier, or as a wetted solid e.g. by pre-wetting the support before mixing it with the choline compound. As is noted above, pre-wetting is particularly suitable for cellulosic carriers, which otherwise may not be

thoroughly wetted by the choline compound solution and may thus not absorb it into the pores of t carrier. After the initial mixing 'in the wet' the choline compound on the carrier can be dried befor or during dispersion in the protective matrix e.g. by heating with the molten fatty acid as describe above. We believe that this approach results in the choline compound being held within the surfa pores of the support and that the fatty acid caps the pores as well as providing a bulk matrix or coating.

In a particularly advantageous aspect of the invention, we have found that by using an alkaline solution of choline compound (as compared with the choline compound solutions of commerce) to make the supported choline compound on the carrier, the protection of the choline compound can be much more effective. We believe that this is because the alkali, which will be present in the pores of the carrier, reacts with the fatty acid to form a soap which caps the pores o the carrier. This appears to be more effective in protecting the choline compound than the fatty a pore caps referred to above. The presence of fatty acid soaps, particularly alkali metal soaps, is further beneficial in that they appear to plasticise the fatty acid matrix. This can make the matrix both less likely to fracture under mechanical working in the gut, enabling a larger proportion of matrix to be usefully employed, and less susceptible to penetration by the contents of the rumen. These effects increase further the effectiveness of the protection of the choline compound from degradation in the rumen.

The amount of alkali needed to achieve this effect (however it works) is not large. We hav obtained good results just by adding sodium hydroxide to raise the pH of choline chloride solution above about 11. Other alkali materials can be used particularly alkali and alkali earth metal hydroxides and carbonates. The molar proportion used will typically correspond to the amount of sodium hydroxide indicated above. We believe that the final pH of the choline compound is not critical (some alkali materials cannot achieve pH's as high as 11). Where the carrier is lime, of course, this is itself alkali and adding alkali to the choline compound solution is not necessary to form a soap pore cap. Nevertheless it may be useful to include alkaline metal alkali to provide th corresponding soap in the fatty acid matrix. This possibility forms a separate and advantageous aspect of the invention.

Accordingly, the invention includes a ruminant feedstuff additive, which comprises a post-ruminally effective choline compound releasably supported on an acceptable earner, coated or dispersed within a protective matrix having a melting point of at least 40° C, the matrix being made up of one or more water insoluble fatty acid(s) and comprising at least 35% by weight of th additive and wherein the choline compound is held in pores in the carrier which pores are capped a fatty acid alkali or alkali earth metal soap within the matrix. Desirably in this aspect of the invention, the fatty acid matrix includes a small proportion of fatty acid soap, particularly an alkali metal fatty acid soap. The alkali metal soap appears to act t plasticise the fatty acid matrix and improve its protective properties as referred to above. Of course, the amount of alkali metal soap included in the matrix will not be so high as to make the matrix sufficiently water soluble or dispersible as to lessen the protection of the choline compoun

We believe that even the small amounts of alkali included in the choline compound solution to adjust its pH in this aspect of the invention can give beneficial plasticising effects.

The amount of water soluble fatty acid soap included in the matrix will usually be at least 0.1% and more usually at least 0.25% by weight of the matrix as lower proportions have very little effect. The proportion of water soluble soap will not usually be more than about 5% by weight of the matrix as such higher levels can make the matrix sufficiently water soluble (even if only by leaching of the soap) that the protection of the choline from the contents of the rumen is reduced. For optimum effect the proportion of such soap will not usually be more than about 2% by weight of the matrix even when relatively high proportions of choline compound are used. For typical choline (as chloride) levels in the feedstuff additive of about 25% by weight, the level of water soluble fatty acid soap will usually be about 1% by weight of the matrix. At lower levels of choline, the weight percentage on the matrix will usually be somewhat lower based on the matrix but may be higher than the 1 :25 ration based on the choline compound indicated above.

In addition to the post-ruminally effective choline compound the feedstuff additive may contain additives for example amino acids, such as methionine, vitamins and minerals which are also desired to be available post-ruminally to the ruminant.

From the figures given above, typical and preferred quantitative compositions of the feedstuff additive are given below:

Material wt % of feedstuff additive typical preferred matrix 35 to 80 50 to 60 supported choline compound 20 to 65 40 to 50 carrier 6 to 40 16 to 30 choline compound 8 to 45 16 to 30

The invention includes feedstuff compositions including the additive of the invention. The additive itself can be included in feed given to ruminants e.g. in the form of pellets mixed with pellets of conventional feedstuff. Alternatively, the additive can be incorporated, along with other feedstuff components, into a uniform feedstuff for feeding to ruminants. A particular end use we envisage for the additive and feedstuffs of this invention, is in enhancing milk yields especially in cows. Generally, in this end use, the optimum amount of choline (as chloride) supplied to a cow is typically about 35 g.day . The amount and or concentration of the additive needed to achieve this, or another desired, feeding rate can readily be calculated. An additional benefit of the additive of this invention, particularly as compared with additives in which the choline is protected with a polymeric coating, is that the protective matrix provides useful nutritional values, particularly as calories. As feedstuffs often include fats and/or oils as components of the feedstuff, the nutritional value of the fatty acid material used as the protective matrix will generally reduce the amount of other fatty materials necessary to provide the desired nutritional values.

The invention is illustrated by the following EΞxamples. All parts and percentages are by weight unless otherwise specified and for compositions are on a dry basis (except for solution concentrations).

In the [Examples the choline was provided as choline chloride in one of the following forms: CC 75% aqueous choline chloride solution. CP corn premix made by mixing crushed com (maize) cob carrier with CC solution and containing about 50% w/w choline chloride. WCP wetted com premix made by mixing pre-wetted crushed com (maize) cob carrier with CC solution and containing about 50% w w choline chloride. SP silica premix made by mixing hydrophilic paniculate silica with CC solution and containing about 50% w/w choline chloride. The silicas used were Tixosil 34A from Rhone Poulenc, Sipemat 22 from Degussa and Hisil HOA from PPG. There was no noticeable difference between the various silicas. ASP silica premix as SP but made with choline chloride solution with the pH adjusted using NaOH to 12.85.

The CP, WCP, SP and ASP premixes were all free flowing powders as the support readily absorbs the choline chloride solution.

In testing some of the compositions as described below a simulated rumen was used. The simulated rumen is a stirred solution of 1M ammonium acetate solution held at 40"C. Despite the apparent simplicity of this model, we have found that it gives results in testing for protection of choline chloride from dissolution into the liquid continuous phase from a feed additive that correspond fairly well with results obtained in vivo in cattle rumens.

Example 1 to 5 and C1 to C4 These examples illustrate the incorporation of choline chloride into fatty acid or fatty acid soap matrix materials. Choline chloride, in one of the forms listed above, was incorporated into a protective matrix of either palmitic acid or the calcium palmitate (soap).

Example C1

Molten palmitic acid (90 g; mp 63°C) in a 250 ml glass beaker was stirred using a 4-blade impeller driven by a Heidolf Laboratory stirrer. CC solution (13.3 g; containing 10 g choline chloride) was added, to give a choline content of 10% by weight of the overall composition. The mixture was heated and stirred to remove free water. During heating the choline chloride formed a separate lower layer, which disappeared on cooling. After cooling, the composition was vacuum dried. The dried composition was very sticky and did not form separable particles. Attempts to grind the dried composition to form definite particles were unsuccessful. Varying the stirring speed from 35 to 250 r.p.m. (ca. 0.6 to ca. 4 Hz) speeded the preparation but did not otherwise alter the form of the

product. It was not practical to test this product further or a similar product made with twice the amount of CC solution.

Example C2

CC solution (16.4 g) was added to palmitic acid (102.4 g) and the mixture heated to melt the acid. Initially the CC solution formed a layer below the molten acid, but the two layers lowly merged on further heating. Calcium hydroxide ( 14.8 g) was added to the mixture. The resulting composition, which had a choline content of 10% w/w, was lumpy and non-uniform in appearance. As in Example C1 , it was not practical to test this material further.

Example C3 Calcium palmitate was prepared by melting palmitic acid (102.4 g) and adding calcium hydroxide (14.8 g) with slow stirring and CC solution (16.4 g) was then added. The CC solution remained separate until heated further, when some of the solution was absorbed. On cooling it was noted that the choline chloride was separated from the calcium palmitate. As in Examples C1 and C2, it was not practical to test this material further.

Example 1

Morten palmitic acid (60 g; mp 63°C) in a 250 ml glass beaker was stirred as described in comparative Example C1. Silica Premix (SP) (40 g; containing 20 g choline chloride) was added, to give a choline content of 20% by weight of the overall composition. The mixture was heated and stirred to remove the water present and was then cooled and vacuum dried. The product was in the form of separate particles with an outside coating of the fatty acid.

Example 2

Example 1 was repeated but substituting an equal weight of com premix (CP) for the silica premix used in Example 1. During cooling some separation of the premix occurred. Apart from this the product appeared much like that of Example 1.

Example 3 a) Silica premix (SP) (74 g) was added to solid palmitic acid (102.4 g) and the mix was heated to melt the acid. Ca(OH), (14.8 g) was then added to convert the acid into the calcium salt. Addition of the calcium hydroxide was difficult because a substantially immobile mass formed. The mixture was heated to remove the water present and then cooled. The resulting composition containing 20% by weight of choline chloride, was non-uniform in appearance. b) Example 3a) was repeated except that the amount of silica premix used was sufficient to give a composition containing 10% by weight of choline. The mixing and appearance of the product were similar to Example 3a.

Example 4

Molten palmitic acid (60 g; mp 63°C) in a 250 ml glass beaker was stirred as described in comparative Example C1. Pre-wetted com premix (WCP) (40 g; containing 20 g choline chloride) was added, to give a choline content of 20% by weight of the overall composition. The mixture was heated and stirred to remove the free water present. The mixture was cooled and vacuum dried. The dried composition was in the form of separate particles with an outside coating of the fatty acid.

Example 5

Example 4 was repeated but using 80 g palmitic acid and 20 g of pre-wetted com premix (WCP).

The product was of similar appearance to that of Example 4.

Example C3 a) Palmitic acid (10 g) was melted in the presence of silica premix (SP) (90 g). After stirring and cooling the resulting composition was uniform and free from lumps b) Example C3a was repeated using 15 g palmitic acid and 85 g silica premix. The product appeared similar to that of Example C3a. c) Example C3a was repeated using 20 g palmitic acid and 80 g silica premix. The product appeared similar to that of Example C3a except that globules were noted on the surface of the composition.

Example C4

Parts a, b and c of Example C4 were repeated but using wetted com premix (WCP) instead of the silica premix in Example C4. The products appeared similar to those of Example C3.

The products of Examples 1 to 5 and comparative Examples C3 and C4 were tested for their ability to protect the choline as follows :

A 0.5 g sample of each composition was dispersed into 50 ml of glacial acetic acid to give a suspension which was titrated until neutral with 0.1 M perchloric acid using a methyl violet indicator. 10 ml of mercuric acetate (6 g in 100 ml glacial acetic acid) was then added. The samples were then stirred for 90 minutes with subsequent retitration using perchloric acid. The amount of choline released into the titration medium was calculated from the titration results and expressed as a percentage loss of choline chloride from the composition.

The compositions and results of testing are set out in Table 1 below. From the results given in Table 1 , it can be seen that palmitic acid can give effective protection to choline chloride supported on a carrier, but that the protection is not effective if the amount of the protective matrix is too small. Also palmitic acid appears to be more effective than calcium palmitate in protecting the choline. The best results were obtained using a wetted com premix and a suitably high proportion of palmitic acid as the protective matrix.

Example C5

A commercially available composition containing 25% w/w choline on a silica support protected with a matrix of a mixture of C ₁₂ to C ₂₂ saturated and unsaturated vegetable fatty acids, having a melting point of less than 40°C was tested in the simulated rumen to measure the rate of dissolution of choline and this was compared with the rate from a comparable, but unprotected, silica premix. The results showing the percentage of choline removed from both the known composition and unprotected premix with time are given in Table 2 below. These results indicate that the commercially available material affords little protection against decomposition in the rumen for the choline in it and that the amount of choline retained within the composition for post-ruminal assimilation was negligible.

Further samples of the commercially available composition were implanted into the rumen of a number of cattle, under veterinary supervision. The choline remaining in the composition after predetermined times in the rumen were measured and choline loss calculated. The loss of choline from the samples with time is set out in Table 3 below. These results confirm that the commercially available composition rapidly looses virtually all its choline in a short time in the rumen and that very little, if any, remains for post-ruminal absorption.

Example 6

A series of compositions were prepared by intimately incorporating a wetted com premix (WCP) into a molten mixture of fatty acids (palmitic acid 45%, stearic acid 40%, oleic acid 10%, C ₁₄ and below - 2%, C2o and above - 3%; melting point about 59 ^β C) as protective matrix. Compositions containing 5, 10 and 50% by weight of the matrix were satisfactorily formed with the corresponding percentage of com premix. Each composition was pelleted by pouring the molten composition into a suitable mould and applying a pressure of 50 MPa until solidification occurred. The structure of the pellets so formed was maintained even during subsequent handling. The dissolution of choline from samples of these pellets was determined by placing pellets of each composition into a simulated rumen. After varying periods of time the amount of choline lost from the pellets was determined. The results are set out in Table 4 below. These data indicate that a protective matrix content of 50% by weight affords substantial protection to the choline present, but that lower levels of fatty acid matrix did not afford effective protection.

Example 7

Compositions were prepared by intimately incorporating 50% by weight of a) silica premix (SP), and b) alkali silica premix (ASP), into 50% by weight of a molten protective matrix of the same mixture of fatty acids used in Example 6. The bulk mixtures were allowed to cool and solidify and were then cut up into ca. 1 cm ³ lumps. Samples of the lumps were tested for their ability to protect the choline in the simulated rumen for 30 and 120 minutes. The percent choline loss is shown in Table 5 below from which the improvement in the protection of the choline is evident.

Example 8

3

Further samples were prepared as cut 1 cm lumps as described in Example 7 based on a) silica premix (SP), and b) alkali silica premix (ASP). A proportion of each sample was ground to a coarse powder having a particle size of ca 1 mm. The four samples were then tested for their ability to protect the choline in the simulated rumen for 120 minutes. The results are given in Table 6 below. These results indicate that the increase in the surface area of the powder samples results in increased choline loss, but the difference between the samples based on ASP show that the difference is small, suggesting that this form of the feedstuff will have increased resistance to mechanical working in the rumen as compared with the samples based on SP.

Table 1

Additive Composition %

Ex Matrix Carrier Choline Choline No Loss

Material % Material % %

C3a Palmitic Acid 10 CP 45 45 95

C3b Palmitic Acid 15 CP 42.5 42.5 91

C3c Palmitic Acid 20 CP 40 40 85

C4a Palmitic Acid 10 SP 45 45 84

C4a Palmitic Acid 15 SP 42.5 42.5 93

C4c Palmitic Acid 20 SP 40 40 83

1 Palmitic Acid 60 SP 20 20 52

2 Palmitic Acid 60 CP 20 20 64

3a Calcium Palmitate 60 CP 20 20 69

3b Calcium Palmitate 80 CP 10 10 68

4 Palmitic Acid 60 WCP 20 20 46

5 Palmitic Acid 80 WCP 10 10 29

Table 2

Time % Removed (minutes) Protected Non- Protected

0 0 0

15 85.5 98.7

30 87.6 98.2

60 89.6 98.2

180 94.8 98.9

360 96.3 99.1

1440 98.9 100

Table 3

Time (minutes) 2 60 150

% Removed 90.9 99.8 99.92

Table

Table 5

Ex No Choline loss (%) after

30 min 120 min

7a 44 48

7b 24 36

Table 6

Ex No Particle size Choline loss (%) after 120 min

8a powder 78 lumps 54

8b powder 36 lumps 33