NZ547717A - An animal supplement and method of manufacture - Google Patents

Abstract

Disclosed is a method of manufacturing a liquid formulation for oral administration to provide an animal with at least one trace element and/or vitamin by the steps of: (a) producing a solution containing: (i) the trace element/s or vitamin/s selected from one or more of the following: at least one water soluble selenium source; at least one water soluble iodine source; ascorbic acid; and (ii) at least one sugar alcohol based non-aqueous liquid to semi-liquid carrier; (b) producing a solution containing at least one vitamin oil selected from: vitamin A, vitamin D, vitamin E, and combinations thereof; wherein the vitamin oil or oils are characterised by being substantially water immiscible; (c) mixing the solution of step (a) with the solution of step (b).

Description

R "*nte^cfuaT7 Office of ~ 1 JUN 2007 £$M!ved PATENTS FORM NO. 5 Fee No. 4: $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No: 547717 Dated: 6 June 2006 AN ANIMAL SUPPLEMENT AND METHOD OF MANUFACTURE We, Ultimate Equine Limited, New Zealand Company of 160 Hepburn Creek Road, Warkworth, New Zealand hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: AN ANIMAL SUPPLEMENT AND METHOD OF MANUFACTURE STATEMENT OF CORRESPONDING APPLICATIONS This application is based on the provisional specification filed in relation to New 5 Zealand Patent Application Number 547717, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD The invention relates to an animal supplement and method of manufacture. More 10 specifically, the invention relates to a supplement formulation to provide an animal with a range of trace elements and vitamins.

BACKGROUND ART Supplements are known in the art to provide animals such as sheep, cattle, horses 15 and the like with nutrition requirements in order to maintain animal health as well as to treat or prevent disease, avoid pests and numerous other health or final product benefits.

A large problem with such supplements is that the individual components of such formulations have differing chemical properties which mean that simple mixing of the 20 raw ingredients may result in a poor efficacy e.g. precipitate formation and/or breakdown of the ingredient active agents. This is especially the case when there is an aqueous component to the formulation.

To solve the above problem, one approach has been to produce formulations that take into account the differing chemical properties of the various ingredients. 25 However, an issue with this is that the resulting formulations tend to be specific to only one or two active ingredients rather than a range of components. The disadvantage of this, besides purchasing cost, is the added amount of labour and time required to administer the various formulations required to meet a range of vitamin and mineral requirements. 2 It should be appreciated that ideally, supplements be easy to administer, preferably with a minimum of labour, and that the supplement be a single dose rather than for example, the farmer having to administer multiple agents to large numbers of animals.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors 10 assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or 20 elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF THE INVENTION According to one aspect of the present invention there is provided a method of manufacturing a liquid formulation for oral administration to provide an animal with at least one trace element and/or vitamin by the steps of: (a) producing a solution containing: 3 (i) at least one trace element; (ii) at least one vitamin; and (iii) at least one non-aqueous liquid to semi-liquid carrier; characterised in that the trace elements or vitamins are water soluble; (b) producing a solution containing at least one vitamin oil wherein the vitamin oil or oils are characterised by being substantially water immiscible; (c) mixing the solution of step (a) with the solution of step (b).

Preferably, the trace elements and/or vitamins are dissolved in the non-aqueous carrier during step (a).

The inventor has found that by completing the above method, it is possible to produce a single formulation that includes various active agents in a stable form.

In one preferred embodiment, the trace elements and vitamins used in step (a) are: at least one selenium source; at least one iodine source; and, ascorbic acid (vitamin C).

It should be appreciated by those skilled in the art that these elements and vitamin tend to be water soluble and also tend to rapidly break down when in aqueous form thus reducing the ingredient efficacy. It is therefore desirable to stabilise these components using the invention method in order to produce a shelf stable and high 20 efficacy product.

Preferably, the selenium source is sodium selenate. Preferably, the sodium selenate mixed with the non-aqueous carrier is a powder with a particle size of less than approximately 100 mesh screen. 4 In the inventor's experience, it is critical that all sodium selenate is dissolved before other components are added. To help achieve this, it is preferable that the sodium selenate is ground into a fine particle size powder before it is added to the nonaqueous carrier substance.

Preferably, the iodine source is sodium iodide. Most preferably, the sodium iodide mixed with the non-aqueous carrier is a powder with a particle size of less than approximately 80 mesh screen.

Preferably, the ascorbic acid mixed with the non-aqueous carrier is a crystalline powder with a particle size of less than approximately 80 mesh screen.

Preferably, the non-aqueous carrier in step (a) and step (d) if used is glycerol. Glycerol is a preferred carrier for its well known pharmaceutical aid properties including the fact that it stabilises the normally water/air unstable elements and/or vitamins used in the formulation. In addition, glycerol is relatively easy to handle, pH neutral and soluble with water which is the preferred carrier for eventual application 15 of the final product.

Preferably, the vitamin oil or oils used in step (b) are selected from vitamin A, vitamin D, vitamin E, and combinations thereof. It should be appreciated that vitamins A, D and E are naturally found as water insoluble oils. Whilst dried vitamin formulations are available, they are less desirable as they are less potent, pure and stable than oil 20 formulations and they also require additional processing and formulation ingredients. Given the advantages of oils, it should be appreciated that the present invention is desirable as it not only uses the natural oils but also presents these oils in a water suspendable form.

Preferably, the solution of step (b) includes at least one emulsifying agent. In one 25 embodiment, the emulsifying agent may be macrogol-glycerol hydroxystearate 40.

In a preferred embodiment, the solution of step (a) is produced by: (i) heating the liquid to semi-liquid non-aqueous carrier to approximately 100°C and then mixing in at least one selenium source; (ii) then reducing the temperature to below approximately 80°C and mixing in at least one iodine source; and, (iii) then reducing the solution temperature to below approximately 60°C and then mixing in ascorbic acid.

Preferably, during each step (i) to (iii) the solution is agitated.

Preferably, additional non-aqueous carrier may be mixed in during any of steps (i) to (iii). This may be done as a means to control the mixture temperature i.e. carrier at room temperature or lower may be added to a hot mixture to decrease the overall mixture temperature. In addition, further carrier may be added to enhance the 10 solubility of each subsequent added powdered ingredient.

Preferably, the solution of step (b) is produced by: (i) mixing the vitamin oils together and then; (ii) mixing in at least one emulsifying agent to the mixture of step (i).

Preferably, the vitamin oils are mixed together in step (b) at a temperature of less 15 than approximately 60°C.

Preferably, the vitamin oils are completely mixed together in step (i) before emulsifying agent is added in step (ii).

Preferably, the emulsifying agent or agents used in step (b) are in a substantially liquid form.

Preferably, during step (c), the solution is maintained at a temperature of approximately 40°C to 60°C while the solution of step (a) is mixed with the solution of step (b).

In preferred embodiments, the method includes further steps (d) and (e) completed before step (c) of: 6 (d) producing a solution containing at least one chromium source with at least one non-aqueous carrier; (e) mixing the solution of step (d) with the solution of step (a) and continuing with step (b) and step (c) where the solution of step (a) used in step (c) also includes the solution of step (d).

Preferably, the chromium source used in step (d) is chromium picolinate. Preferably, when mixed with the non-aqueous carrier the chromium picolinate is a crystalline powder with a particle size of less than approximately 200 mesh screen. It is the inventor's experience that a very fine particle size is preferable to ensure full mixing.

Preferably, addition of each trace element and/or vitamin into solution during steps (a), (c) and (e) if present is not completed until each trace element compound or vitamin is fully dissolved.

Preferably, the non-aqueous carrier in step (d) is glycerol.

In particular, the inventor has found that the chromium solution of step (d) if used, 15 should not be added to the mixture from step (a) during step (e) until it is certain that all of the chromium source is dissolved in step (d).

Ideally, during step (d), the non-aqueous solution is heated to at least approximately 100°C and held at this temperature while the chromium source is mixed in step (d). More preferably, the temperature is equal to or greater than 125°C. This higher 20 temperature is preferably as the chromium picolinate will only completely dissolve in glycerol at this elevated temperature. It should be appreciated that this higher temperature may result in degradation of other active agents such as vitamins and other trace elements. As a result step (d) should be completed separate to other steps and the resulting mixture of chromium picolinate and glycerol should be cooled 25 before addition to other agents in step (e).

Preferably, agitation occurs during steps (d) and (e). Agitation is considered a critical step based on the inventors experience otherwise insufficient mixing occurs. 7 Preferably, the solution of step (d) is cooled to less than 60°C before step (e) is completed.

It is the inventor's experience that it is very important to keep the steps in exact order. The powders must be dissolved completely and in proper sequence or the 5 final product will not form a complete mixture and elements or vitamins may separate out of the solution.

It is noted that the temperatures and times provided above are given by way of example only. It should be appreciated that the temperatures and times provided may be varied without departing from the scope of the invention.

Preferably, during step (c), irrespective of whether or not steps (d) and (e) are completed, mixing is complete when the mixture of step (c) has a consistent colour and there are no lines of other colour or shine/texture on the mixture surface. The inventor has found this visual indicator is useful in confirming that complete mixing has occurred and occurs as the solutions of steps (a) (and (d) if used), have a 15 different colour to that of step (b).

Preferably, the liquid formulation is mixed with / suspended in water before administration to an animal. It is preferable to produce the above formulation in concentrated form for storage and handling and then dilute / suspend the formulation in water prior to administration. This is to minimise storage size and transportation 20 costs. In one preferred embodiment the liquid formulation is mixed with water at a ratio of 1 part formulation to 4 parts water although it should be appreciated that this ratio may vary depending on the concentration of the initial formulation and concentration to be administered. Dosage may also vary depending on the bodyweight of the animal.

According to a further aspect of the present invention there is provided a liquid formulation for oral administration produced by the method substantially as described above. 8 Preferably the formulation is formulated for oral administration to a ruminant animal.

According to a further aspect of the present invention there is provided a liquid formulation for oral administration to an animal containing: at least one trace element and/or at least one vitamin that is water soluble; at least one vitamin oil that is characterised by being substantially immiscible in water; and at least one liquid to semi-liquid non-aqueous carrier.

According to a further aspect of the present invention there is provided a liquid formulation for oral administration to an animal containing: at least one trace element and/or at least one vitamin that is water soluble; at least one vitamin oil that is characterised by being substantially immiscible in water; and at least one liquid to semi-liquid non-aqueous carrier; characterised in that the formulation is stable at room temperature for at least one month when stored in the absence of moisture and light.

According to a further aspect of the present invention there is provided a liquid formulation for oral administration to an animal containing: at least one trace element and/or at least one vitamin that is water soluble; at least one vitamin oil that is characterised by being substantially immiscible in water; and at least one liquid to semi-liquid non-aqueous carrier; 9 characterised in that the formulation is stable for at least 72 hours when mixed with water and held at room temperature.

Preferably, the term 'stable' when used above refers to the trace element and/or vitamin levels remaining within 10% concentration by weight of initial levels after 5 storage under conditions as indicated.

Preferably, the term 'stable' also encompasses the formulation not showing any discolouration, separation and precipitate formation over a given time period.

To maintain the efficacy and activity of the resulting product, it is the inventor's experience that it is important to keep the formulation free of moisture and light as much as is practical during storage. It is envisaged by the inventor that bottling of the final product would occur shortly after mixing is complete and that other precautions be taken such as packaging the product in dark and moisture proof containers and use of a dark cover over mixing tanks used in the process in order to minimise light and moisture contamination.

In the inventor's experience, the mixture also remains stable and efficacious for at least 72 hours after mixing with water. In one trial completed by the inventor, stability remained for 40 days in water. By contrast, without stabilisation using the method described above, the active agents would rapidly degrade, well within 72 hours, especially in the presence of light.

Preferably, the trace elements and vitamins used are selected from: (i) at least one selenium source; (ii) at least one iodine source; (iii) at least one chromium source; (iv) ascorbic acid (vitamin C); (v) vitamin A; (vi) vitamin D; (vii) vitamin E; (viii) and combinations thereof.

Preferably, the selenium source is sodium selenate.

Preferably, the iodine source is sodium iodide.

Preferably, the chromium source is chromium picolinate.

Preferably, the ascorbic acid is in the form of a crystalline dry powder.

Preferably, the non-aqueous carrier is glycerol.

Preferably, the formulation includes at least one emulsifying agent.

Preferably, the formulation is diluted with water and administered orally to the animal.

In one preferred embodiment the liquid formulation is mixed with water at a ratio of 1 part formulation to 4 parts water although it should be appreciated that this ratio may vary depending on the concentration of the initial formulation and concentration to be administered. The ratio also varies depending on the animal bodyweight.

Preferably, the animal is a ruminant. More preferably, the animal is selected from: 15 sheep, cattle, and deer. In a preferred embodiment, the animal is a dairy cow.

According to a further aspect of the present invention, there is provided a method of supplementing the nutritional requirements of an animal by oral administration of a formulation substantially as described above.

According to a further aspect of the present invention, there is provided a method of 20 correcting a nutrient deficiency in at least one trace element and/or at least one vitamin in an animal by oral administration of a formulation substantially as described above. 11 According to a further aspect of the present invention, there is provided a method of maintaining animal health by oral administration of a formulation substantially as described above.

According to a further aspect of the present invention, there is provided a method of 5 improving the reproductive performance of an animal by oral administration of a formulation substantially as described above.

According to a further aspect of the present invention, there is provided the use of at least one trace element and/or at least one vitamin in the manufacture of a formulation substantially as described above for supplementing the nutritional 10 requirements of an animal.

According to a further aspect of the present invention, there is provided the use of at least one trace element and/or at least one vitamin in the manufacture of a formulation substantially as described above for correcting a nutrient deficiency in at least one trace element and/or at least one vitamin in an animal.

According to a further aspect of the present invention, there is provided the use of at least one trace element and/or at least one vitamin in the manufacture of a formulation substantially as described above for maintaining animal health.

According to a further aspect of the present invention, there is provided the use of at least one trace element and/or at least one vitamin in the manufacture of a 20 formulation substantially as described above for improving the reproductive performance of an animal.

It should be appreciated from the above description that there is provided an animal supplement formulation, method of manufacture, and various methods of treatment using the formulation that addresses the differing chemistry of the active agents so 25 that the supplement is produced in a form that is stable and homogeneous. As the formulation is able to be dissolved and/or suspended in water, the solution is ideally suited for oral administration. Also, as the formulation combines various agents, it 12 may be applied as single dose rather than having to apply the various agents individually. Further, owing to the stabilised nature of the formulation, even when mixed / suspended with water, the active agents remain stable for a longer time period than would be the case if the agents were not in the above formulation.

BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 shows a graph indicating cumulative proportion of cows having conceived by days after the planned start of the seasonal breeding programme; Figure 2a shows a graph indicating interval from planned start of mating to second service (days) by age group (b) and by treatment group (a); Figure 2b shows a graph indicating interval from planned start of mating to second service (days) by age group (b) and by treatment group (a); Figure 3 Figure 4 shows a graph indicating the estimated marginal mean (and standard of error of the mean) milk solids production (kg milksolids/cow/day) at herd tests 1 to 4 during lactation by treatment; and, shows a graph indicating the estimated marginal mean (and standard error of the mean) log™ SCC (x1000 cells/ml) at herd tests 1 to 4 in the 2003/4 lactation by treatment. 13 BEST MODES FOR CARRYING OUT THE INVENTION The invention is now described in terms of best methods of manufacture known to the inventor. Animal trials are also described confirming efficacy of the formulation. This should not be seen as limiting as it will be appreciated from the description 5 below that variations may be made from the method described without departing from the scope of the invention.

EXAMPLE 1 This Example describes one preferred best method of manufacturing the formulation.

The preferred method of manufacture is by producing three separate solutions in stainless steel mixing tanks each containing different components in the formulation. The three solutions are then mixed together to form the eventual product. It should be appreciated that only two solutions may be required where the range of trace elements and vitamins are varied.

The method below describes quantities sufficient to produce an approximately 2000 litre batch of formulation. It should be appreciated that quantities are provided by way of example only and may be varied as required to increase or decrease the batch size, increase or decrease the amount of an agent and so on.

Producing a Solution Containing Selenium, Iodine and Ascorbic Acid In a mixing tank, add 500 litres of food grade glycerol and heat to approximately 100°C.

Add 5 kg of very finely ground drench grade sodium selenate and strongly agitate the resulting mixture until the sodium selenate is fully dissolved. In the inventors experience this takes approximately 2 hours. During this mixing stage, agitation 25 should be maintained but not to the extent that the solution becomes foamy. The temperature is also maintained at approximately 100°C.

Once the sodium selenate has dissolved, add a further 100L to 200L of food grade glycerol to the tank and reduce the temperature to below 80°C. 14 Once additional glycerol is added, add 240kg of sodium iodide powder whilst the mixture is agitated. Mixing should be continued until the solution is clear of powder (in the inventor's experience this may take at least 1 hour after addition of sodium iodide).

Ideally the sodium iodide powder used is free flowing and free of lumps. In the inventor's experience, this greatly aids in the process of mixing.

Add a further 100 litres to 200 litres of food grade glycerol and reduce the mixture temperature to below 60°C. At this point add 60 kg of fine ascorbic acid (vitamin C) powder. Agitate until the ascorbic acid powder is completely dissolved (at least one 10 hour in the inventor's experience).

Producing a Solution Containing Chromium Heat 400 litres of food grade glycerol to 130°C in a tank. It is the inventor's experience that this temperature is important to ensure complete mixing.

Add 17kg of crystalline chromium picolinate powder and mix well. It is the inventor's 15 experience that this mixing process takes approximately 30 minutes.

Producing a Solution Containing Vitamin Oils In a tank add 200 kg of 1000 i.u./g Vitamin E oil, 20 kg of 1 million i.u. /g Vitamin A oil and 10 kg of 1 million i.u./g Vitamin D oil. Blend the oil mixture together and also heat to approximately 55°C while blending.

Add 160kg of emulsifying agent (in substantially liquid form) and continue blending for approximately 15 to 30 minutes.

Producing the Combined Formulation For process efficiency, this combining step is only completed once the chromium and vitamin oil formulations have been produced and the selenium/iodine/ascorbic acid 25 solution is cooled to less than 60°C.

Add the hot dissolved chromium picolinate solution at 100°C to 125°C to the mixture containing the dissolved sodium selenate, sodium iodide and ascorbic acid. Mix and allow the resulting mixture to cool to between approximately 40°C to 60°C.

Subsequently transfer the warm mixture of vitamin oils and emulsifier into the mixture of chromium picolinate solution and sodium selenate, sodium iodide and ascorbic acid. Continue blending to ensure complete mixing. In the inventor's experience this takes approximately 30 minutes.

It is the inventor's experience that it is important to keep the steps in exact order. The powders must be dissolved completely and in proper sequence or the final product will not form a complete mixture and elements or vitamins may separate out of the solution.

To maintain the efficacy and activity of the resulting product, it is important to keep 10 the product free of moisture and light as much as is practical. It is envisaged that bottling of the final product would occur shortly after mixing is complete and that other precautions be taken such as packaging the product in dark and moisture proof containers and use of a dark cover on the mixing tank in order to minimize light and moisture contamination. If necessary, the final combined solution may be held for 15 over 24 hours before further processing. If stored, the temperature should be kept above 30°C to avoid any degradation.

In the inventor's experience, it is critical that all sodium selenate is dissolved before other components are added. To help achieve this, it is preferable that the sodium selenate is ground into a very fine powder before adding to the glycerol mixture.

A further critical step is that the glycerol temperature when mixing the chromium picolinate with glycerol ideally remains above 120°C at all times to dissolve all the powder but, should also not exceed 130°C.

In addition, the inventor has found that the chromium and glycerol solution should not be added to the sodium selenate, sodium iodide and ascorbic acid mixture until it is 25 certain that all the chromium has dissolved. Ideally the chromium solution mixture is added to the sodium selenate, sodium iodide and ascorbic acid mixture at or above 100°C or precipitation may occur.

One visual indicator of successful blending that the inventor has noted is that the mixture containing the chromium and glycerol solution with the sodium selenate, 30 sodium iodide and ascorbic acid solution has a different colour and shine to the vitamin oil and emulsifier solution. Due to this difference, a visual cue when completing the final mixing step is when the two mixtures have a consistent colour 16 and there are no lines of other colour or shine on the mixture surface. In this Example, the combined mixture colour is a ruby red / opalescent colour.

From the above example, it should be appreciated that there is provided a method and formulation that is non-aqueous and may be produced to contain a variety of 5 trace elements and vitamins. The method and formulation assist to stabilise the active agents and avoid loss in efficacy.

EXAMPLE 2 This example describes an animal study undertaken to confirm efficacy of the 10 formulation.

Methodology A single dairy herd of approximately 200 cows in the Marlborough region of New Zealand was selected for study. The herd calved between July and October with the planned start of the seasonal breeding programme being in October each year. 15 Breeding continued for 60 to 70 days. No induction of parturition or treatment of anoestrous cows was undertaken before or during the study. Milk production and composition was assessed during lactation.

Data for the herd was obtained and cows were blocked by breed (i.e. >11/16th Friesian = F, >11/16ths Jersey =J, rest = crossbred), age code (i.e. 2, 3, >3 years) 20 and then sorted on average somatic cell count (SCC) from herd tests during lactation and then assigned to treatment or untreated control within sequential pairs. Treated cows were orally administered the formulation of Example 1.

At the end of the second calving period (October) data for the cows was again obtained including actual calving data, age, breed, lifetime identification, milk 25 production data, all service dates and any disease data.

Cows included in analysis were those assigned to treatment, that calved during the 17 trial time period (June to October), and that survived until the planned start of the seasonal breeding programme (late October). This left 193 cow records to be analysed. Of these 11 died between 167 and 281 days postpartum of bloat (n=8), being cast (n = 1) and of unknown causes (n=2).

Analysis The Day 21 submission rate, Day 28 and Day 56 pregnancy rate and final pregnancy rate were analysed. Initially univariate (X2 for categorical variables and binary logistic regression analysis for continuous variables) analysis was used and any variable associated (i.e. P<0.2) was offered to reverse stepwise model using likelihood ratio 10 as the inclusion/exclusion approach. Independent variables included treatment, age code (2, 3, 4+ years), breed code (J, F and XB) and occurrence of clinical mastitis in the current season (yes/no), postpartum interval (i.e. interval from calving to start of seasonal breeding programme as a continuous variable in days) and breeding worth. Treatment was always included in the models. The model validity was assessed by 15 assessing the Hosmer-Lemeshow 'goodness of fit' and assessing that the number of outliers as < 5%.

The interval between the start of the seasonal breeding programme and first service and conception were initially analysed using Kaplan-Meier survival analysis and then with Cox's proportion hazards models.

The interval from the planned start of the seasonal breeding programme to the second recoded insemination was analysed by Kaplan-Meier survival analysis. Only those cows not conceiving to the first insemination were included in this analysis.

Milk production and somatic cell count (SCC) data were analysed using a repeated measures analysis of variance model. The main effects included were treatment, age 25 group (coded as 2, 3 and 4+ years), breed (coded as Friesian, Jersey or crossbred), mastitis history in the current lactation (yes/no) and calving date was included as a 18 covariate. All first order interaction between treatment and the other variables were tested. In all cases the assumptions of sphericity were violated so Greenhouse-Giesser tests were used to assess significance.

Results The treatment groups had similar age, calving date, breeding worth, milk volume and somatic cell counts (SCC) at herd tests 1 to 3 in the season before treatment (Table 1). However, the treated group had higher milk volume production at herd tests 1 to 3 before treatment (Table 1).

Table 1 - Descriptive pre-treatment (i.e. milksolids (MS), milk volume (Vol) and somatic cell 10 count (SCC) at herd tests (HT) 1 to 3 in the previous lactation) data for cows treated with formulation (Treated) or left as untreated controls. (P=univariate P value).

Control Treated N Mean SD N Mean SD P Age years 99 4.9 2.6 94 4.8 2.2 0.934 Calving date 99 16/8 14.8 92 17/8 17.7 0.606 Breeding worth 99 120.1 .5 94 124.7 29.1 0.282 MS HT1 pre kg/cow/ day 78 1.63 0.31 71 1.74 0.40 0.049 MS HT2 pre kg/cow/ day 80 1.64 0.29 77 1.72 0.31 0.078 MS HT3 pre kg/cow/ day 80 0.96 0.17 77 1.01 0.16 0.074 Vol HT1 pre L/cow/ day 78 18.3 4.3 71 19.4 .3 0.171 Vol HT2 pre L/cow/ day 80 16.6 3.7 77 17.2 4.0 0.289 Vol HT3 pre L/cow/ day 80 9.7 2.0 77 .2 2.1 0.145 SCC HT1 pre x1000 cells/mL 78 89 131 71 97 188 0.772 SCC HT2 pre x1000 cells/mL 80 121 197 77 119 187 0.949 SCC HT3 pre x1000 79 221 19 333 77 193 210 0.530 cells/mL Ave SCC pre x1000 80 144 186 77 137 171 0.810 cells/mL Max SCC pre x1000 80 239 353 77 217 255 0.668 cells/mL Reproduction The Day 21 submission rate was unaffected by treatment (RR = 1.02 (0.96-1.04); p = 0.41; Table 2).

Table 2 - Descriptive analysis of reproductive performance of cows left untreated (Control) or 5 treated (Treated) with formulation. P values are the univariate probabilities of difference between groups (i.e. X2 for dichotomous variables and Kaplan-Meier survival analysis for the continuous variables (Median and SEM)).

Control Treated P Variable No.

No. total % No.

No. total % Oestrus D21 92 99 92.9 90 94 95.7 0.40 Oestrus D28 97 99 98.0 92 94 97.9 0.96 Conceive to 1 st service 37 90 41.1 43 86 50.0 0.24 2nd service1 32 53 60.4 43 58.1 0.82 Pregnant D28 45 87 51.7 56 84 66.7 0.05 Pregnant D56 62 87 71.3 73 84 86.9 0.01 Not pregnant (final) 19 98 19.4 7 94 7.4 0.02 PSM2 to 1 st service (days) 1 9 1 0.79 PSM2 to conception (days) 26 4 19 1 0.01 The probability of conception to first insemination tended to be increased by treatment (RR = 1.25 (0.95-1.51); P = 0.10; Table 2) and was lower in cows calved 10 <42 days at the planned start of the seasonal breeding programme (4/21 (19.0%) vs. 76/153 (49.7%) 1st service conception rate for cows calved <42 days vs. cows calved 42 + days at the start of the seasonal breeding programme (FR = 0.35 (0.13-0.81); P = 0.01)).

A similar proportion of cows that had not conceived to first insemination were inseminated for a second time between treatments (Table 2). The interval from the start of the seasonal breeding programme to the second service was not different between groups (29 (4) vs. 36 (10) median (SEM) days from PSM to 2nd service for 5 treated vs. control cows, respectively; P = 0.65); Figure 2a). However, 3-year-old cows were less likely to have second insemination than 2- or 4+ year-old-cows (Figure 2b; P = 0.01).

The Day 28 pregnancy rate tended to be increased by treatment (RR =1.17 (0.98-1.30); F = 0.07), and tended to be positively related to breeding worth 10 (RR = 1.01(1.00-1.02), P = 0.07; Table 2).

The Day 56 pregnancy rate was increased by treatment (RR = 1.08 (1.01-1.12); P = 003) and was positively related to breeding worth (RR = 1.02 (1.01-1.04), F = 0.001; Table 2).

The final pregnancy rate was increased by treatment (RR 1.05 (1.01-1.07); 15 P = 0,03) and was positively related to breeding worth (RR = 1.01 (1.00-1.03), P = 0.06; Table 2).

The median interval to first insemination did not differ between groups (11 (1) vs. 11 (1) mean and (SEM) for control and treated cows respectively; P = 0.78; Table 2).

The median interval from the start of the seasonal breeding programme to pregnancy was shorter for treated than control cows (19 (1) vs. 26 (3) median (SEM) days from the start of the seasonal breeding programme to conception for treated and control cows, respectively; P = 0.78; Figure 1). The hazard of conception was increased by treatment (HR =1.60 (1.14-2.24), P = 0.007) and increased with increasing interval 25 from calving to PSM (HR = 1.01 (1.00-1.02), P = 0.01). 21 Milk Production and Quality Milk solids production was not affected by treatment (1.53 ± 0.03 vs. 1.51 ± 0.03 mean ± SEM milk solids production (kg/cow/day) for treated and control animals respectively; P = 0.319) and there was no time by treatment interaction (P 0.691; Table 3; Figure 3). Milk production varied among age groups (1.35 ± 0.04, 1.51 ± 0.04 and 1.70 ± 0.03 mean ± SEM milk solids production (kg/cow/day) for 2-year-olds, 3-year-olds and 4+ year olds, respectively; P<0.001) and breeds (1.62 ± 0.05, 1.58 ± 0.04, and 1.37 ± 0.02 mean ± SEM milk solids production (kg/cow/day) for Friesians, Crossbreds and Jerseys, respectively; P<0.001) and tended to be higher in cows diagnosed with mastitis (1.56 ± 0.04 vs. 1.48 ± 0.02 mean ± SEM milk solids production (kg/cow/day) for cows diagnosed with mastitis vs. not diagnosed with mastitis, P= 0.07).

Table 3 - Final repeated measures ANOVA between subjects analysis for milk solids product Source Type III Sum of Squares df Mean Square F Sig.

Intercept 88.2 1 88.2 681.5 0.000 Treatment 0.1 1 0.1 1.0 0.319 Age group 12.8 2 6.4 49.6 0.000 Breed 6.1 2 3.0 23.6 0.000 Mastitis (03/04) 0.4 1 0.4 3.4 0.068 Postpartum interval 0.2 1 0.2 1.5 0.220 Error 22.3 172 0.1 Logi0 SCC was not affected by treatment (2.04 ± 0.05 vs. 2.06 ± 0.05 mean ± SEM log10SCC (x 1000 cells/ml) for treated and control animals respectively; P = 0.72) and there was no time by treatment interaction (P = 0.77; Table 4; Figure 4). Milk production varied among age groups (1.93 ± 0.06,1.99 ± 0.08 and 2.22 ± 0.05 mean ± SEM log10 SCC for 2-year-olds, 3 year-olds and 4± year olds, respectively; P<0.001) and was higher in cows diagnosed with mastitis (2.12 ± 0.07 vs. 1.97 ± 22 0.04 mean ± SEM logic SCC for cows diagnosed with mastitis vs. not diagnosed with mastitis, P = 0.04).

Table 4 - Final repeated measures ANOVA between subjects analysis for log 10 somatic cell count (x1000 cells/ml).

Source Type III Sum of Squares df Mean Square F S/flf.

Intercept 152.1 1 152.1 364.2 0.000 Treatment 0.1 1 0.1 0.1 0.722 Age group 11.0 2 .5 13.1 0.000 Breed 0.1 2 0.0 0.1 0.916 Mastitis (03/04) 1.8 1 1.8 4.2 0.041 Postpartum interval 0.2 1 0.2 0.5 0.501 Error 71.4 171 0.4 There was no difference in the number of cows diagnosed with clinical mastitis in the trials between treatments (16/99 (16.2%) vs. 9/94 (9.6%) for control and treated cows, respectively; P = 0.173).

Trial Summary Cows dosed with the formulation of the present invention showed significantly 10 improved reproductive performance following supplementation. Treatment with the formulation resulted in improved pregnancy rates compared to control groups with increased conception rates during the trial time period.

Inspection of the cumulative pregnancy rate data suggests that the performance of treated animals diverged approximately 2 weeks after the planned start of the 15 seasonal breeding programme with treated cows having better reproductive performance than control cows after this time period.

Whilst not fully understood, it is considered that a key reason for the observed improved reproductive performance was the improved health and dietary supplementation cows received from the formulation. 23 No negative effects were observed by administration of the formulation of the present invention on the cow. Milk solids production and infection rates as measured via SCC was also not negatively influenced by administration.

EXAMPLE 3 The stability of the formulation of Example 1 was tested.

In a first test, the formulation was stored in packaging to eliminate exposure of the formulation to light and to moisture. The packaged formulation was stored at ambient conditions for approximately one month and subsequently tested. The 10 concentration of trace elements and vitamins measured after storage was compared against that measured from a sample taken before storage. The variation observed between the two tests was a less than 10% variation between before and after storage. This test confirmed the stability of the formulation during storage.

In a second test, the formulation was placed in a container with water at a 1:4 dilution 15 and agitated. The formulation and water combination was initially tested and then sampled again after 25 days and 40 days at room temperature and in dark conditions. Samples were analysed to compare the stability of selected trace element and vitamin levels over time. Again, the formulation showed a strong stability under trying conditions (exposure to water) and the variation observed 20 between the two tests was for most components a less than 10% variation between before and after storage as shown below in Table 5. Vitamin C showed a greater decrease. To rectify this loss the drench may be reformulated to include more Vitamin C.

Table 5 - Storage Stability of the Formulation in Solution over Time Vitamin/Mineral Initial Level Days Storage 40 Days Storage Vitamin A 1270lU/ml 1290 1350 24 Vitamin D 1180 Ill/ml 1120 1060 Vitamin E 17.8 lU/ml 18.1 17.5 Vitamin C 418 mg/100ml 171 96.9 Selenium 200 mg/kg 187 214 This test further confirmed the stability of the formulation after storage.

In a third test, the formulation was placed in a container with water and agitated as in the second test but left exposed to the light. The combination was initially tested for vitamin E and iodine levels and then left for 22 and 74 hours and subsequent 5 samples taken. After this time period there was no loss in concentration of vitamin E or iodine levels. This test further confirmed the stability of the formulation after storage.

From the above Examples it should be appreciated that there is provided a single dose formulation for delivery of trace elements and vitamins. The manufacturing 10 method overcomes the differing chemical properties of the trace elements and vitamins therefore allowing a single dose formulation rather than having to dose an animal multiple times with different formulations of trace elements and vitamins. The animal trial completed confirmed the efficacy of the formulation in maintaining animal health and in particular showed a significant improvement in reproductive 15 performance. The stability tests showed that the formulation remains stable during storage and even under difficult conditions of water exposure, maintains stability over at least 72 hours, and even after 2 weeks.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without 20 departing from the scope thereof as defined in the appended claims.

Claims (36)

WHAT WE CLAIM IS:

1. A method of manufacturing a liquid formulation for oral administration to provide an animal with at least one trace element and/or vitamin by the steps of: (a) producing a solution containing: (i) the trace element/s or vitamin/s selected from one or more of the following: at least one water soluble selenium source; at least one water soluble iodine source; ascorbic acid; and (ii) at least one sugar alcohol based non-aqueous liquid to semi-liquid carrier; (b) producing a solution containing at least one vitamin oil selected from: vitamin A, vitamin D, vitamin E, and combinations thereof; wherein the vitamin oil or oils are characterised by being substantially water immiscible; (c) mixing the solution of step (a) with the solution of step (b).

2. The method as claimed in claim 1 wherein the selenium source is sodium selenate.

3. The method as claimed in claim 2 wherein the sodium selenate mixed with the non-aqueous carrier is a powder with a particle size of less than approximately 100 mesh screen.

4. The method as claimed in claim 1 wherein the iodine source is sodium iodide.

5. The method as claimed in claim 4 wherein the sodium iodide mixed with the nonaqueous carrier is a powder with a particle size of less than approximately 80 mesh screen.

6. The method as claimed in claim 1 wherein the ascorbic acid mixed with the nonaqueous carrier is a powder with a particle size of less than approximately 80 mesh screen. ■ ' APR 2008 ■^cejved 26

7. The method as claimed in any one of the above claims wherein the non-aqueous carrier in step (a) is glycerol.

8. The method as claimed in any one of the above claims wherein the solution of step (b) also includes at least one emulsifying agent.

9. The method as claimed in claim 8 wherein the emulsifying agent is macrogol-glycerol hydroxystearate 40.

10. The method as claimed in any one of the above claims wherein the solution of step (a) is produced by: (i) heating the liquid to semi-liquid non-aqueous carrier to approximately 100°C and then mixing in at least one selenium source; (ii) then reducing the temperature to below approximately 80°C and mixing in at least one iodine source; and, (iii) then reducing the solution temperature to below approximately 60°C and then mixing in ascorbic acid.

11. The method as claimed in claim 10 wherein, during each step (i) to (iii) the solution is agitated.

12. The method as claimed in claim 10 or claim 11 wherein additional non-aqueous carrier may be mixed in during any of steps (i) to (iii).

13. The method as claimed in any one of the above claims wherein the solution of step (b) is produced by: (i) mixing the vitamin oils together and then; (ii) mixing in at least one emulsifying agent to the mixture of step (i). 27 Intellectual Property Office of N.Z. "8 FEB2008 RECEIVED

14. The method as claimed in claim 13 wherein the vitamin oils are mixed together in step (b) at a temperature of less than approximately 60°C.

15. The method as claimed in claim 13 or claim 14 wherein the emulsifying agent or agents used in step (b) are in a substantially liquid form.

16. The method as claimed in any one of the above claims wherein, during step (c), the solution is maintained at a temperature of approximately 40°C to 60°C while the solution of step (a) is mixed with the solution of step (b).

17. The method as claimed in any one of the above claims wherein the method includes further steps (d) and (e) completed before step (c) of: (d) producing a solution containing at least one chromium source with at least one non-aqueous carrier; (e) mixing the solution of step (d) with the solution of step (a) and continuing with step (b) and step (c) where the solution of step (a) used in step (c) also includes the solution of step (d).

18. The method as claimed in claim 17, wherein the chromium source used in step (d) is chromium picolinate.

19. The method as claimed in claim 18 wherein the chromium picolinate mixed with the non-aqueous carrier is a crystalline powder with a particle size of less than approximately 200 mesh screen.

20. The method as claimed in any one of claims 17 to 19 wherein the non-aqueous carrier in step (d) is glycerol.

21. The method as claimed in any one of claims 17 to 20 wherein agitation occurs during mixing in steps (d) and (e). 28 Intellectual Property Office of N.Z. "8 FEB2008 RECEIVED

22. The method as claimed in any one of claims 17 to 21 wherein during step (d), the non-aqueous solution is heated to at least approximately 100°C and held at this temperature while the chromium source is mixed in step (d).

23. The method as claimed in any one of claims 17 to 21 wherein during step (d), the non-aqueous solution is heated to at least approximately 125°C and held at this temperature while the chromium source is mixed in step (d).

24. The method as claimed in any one of claims 17 to 23 wherein the solution of step (d) is cooled to less than 60°C before step (e) is completed.

25. The method as claimed in any one of the above claims wherein during step (c), mixing is complete when the mixture of step (c) has a consistent colour and there are no lines of other colour or shine/texture on the mixture surface.

26. The method as claimed in any one of the above claims wherein the liquid formulation is mixed with / suspended in water before administration to an animal.

27. The method as claimed in claim 26 wherein the liquid formulation is mixed with / suspended in water at a ratio of 1 part formulation to 4 parts water.

28. A liquid formulation for oral administration produced by the method as claimed in any one of claims 1 to 27.

29. The liquid formulation as claimed in claim 28 wherein the formulation is formulated for oral administration to a ruminant animal.

30. A method of supplementing the nutritional requirements of a non-human animal by oral administration of a formulation manufactured by the method as claimed in any one of claims 1 to 27. 29 Intellectual Property Office of N.Z. - 8 FEB 2008 RECEIVED

31. A method of correcting a nutrient deficiency in at least one trace element and/or at least one vitamin in a non-human animal by oral administration of a formulation manufactured by the method as claimed in any one of claims 1 to 27.

32. A method of maintaining non-human animal health by oral administration of a formulation manufactured by the method as claimed in any one of claims 1 to 27.

33. A method of improving the reproductive performance of a non-human animal by oral administration of a formulation manufactured by the method as claimed in any one of claims 1 to 27.

34. A method of manufacturing a liquid formulation for oral administration to provide an animal with at least one trace element and/or at least one vitamin by the steps substantially as described herein with reference to Example 1.

35. A liquid formulation for oral administration to an animal containing at least one trace element and/or at least one vitamin substantially as described herein with reference to Example 1.

36. A method of providing a non-human animal with at least one trace element and/or vitamin using a liquid formulation for oral administration to a non-human animal containing at least one trace element and/or at least one vitamin substantially as described herein with reference to Example 2. Ultimate Equine Limited by their Attorneys JAMES & WELLS Intellectual Property Office of N.Z. - 8 FEB 2008