ANIMAL PRODUCTION

TECHNICAL FIELD

The present invention relates to a method of increasing animal production. Particularly, although not exclusively, the present invention relates to chemical inhibitors and animal production.

BACKGROUND ART

One of the most important considerations surrounding livestock is to increase animal production such as milk, wool and live-weight gain.

A number of methods have been used in the industry to increase animal production.

Hormone-dependent sex differences in growth rate have been known for a long time. It has also been known that growth rate and FCE (Feed Conversion Efficiency) are higher in intact males than in castrates. It was a natural progression with the availability of hormones and other natural or synthetic substances displaying hormone activity which led to experiments aiming at their use to increase production.

Beginning in the mid-1950's, DES (diethyl stilboestrol) and hexoestrol were administered to cattle increasingly in the US and the UK respectively, either as feed additives or as implants, and other types of substances also gradually became available.

In general, such treatment has resulted in 10-15% increases in daily gains, similar improvements in FCE and improvement of carcass quality (increased lean/fat ratio).

Another example of hormone control in animal production are studies of prolactin. This is a hormone of the anterior pituitary whose secretion varies seasonally in many species, has previously been implicated in the control of hair growth in various species including wool growth cycles in primitive and shedding breeds of sheep.

Prolactin receptors have been identified in the wool follicle revealing a physiological mechanism whereby circulatory prolactin can mediate wool growth cycles.

While the use of hormonally active substances in animal production rose, a disadvantage of this animal production means is the opposition to the use which also has increased, because of the theoretical possibility that residues in edible tissues might endanger consumers.

These factors led to the ban in DES in the US, first imposed in 1973. Several reports confirm that DES endangers the health of animal and humans, when repeatedly used in large doses.

In all the animal industries, one of the primary reasons for the use of antibiotic drugs is for production enhancement (increased growth rate and efficiency of feed use). A significant disadvantage of antibiotic use is that resistance to antibiotics can build up, not only in the animal microflora, but also in humans as residual amounts of antibiotics may enter the food chain.. For this reason, dairy cows treated with antibiotics are required to have their milk withheld for a considerable time, resulting in potential economic penalties for the producer..

Other studies that have been conducted to increase animal production include attempts to increase utilisation of protein from animal feeds.

It was thought that forages containing protected protein (proteins that are protected from rumen digestion) would help more protein uptake up by the animals. The

theory was that the animals should grow faster.

Condensed tannins have increased animal performance by increasing the flow of non-ammonia nitrogen into intestine of ruminants. See Want, Y., Douglas, G. B., Waghorn, G. C, Barry, T. N., and Foote, A.G. 1996. Effect of condensed tannins in Lotus corniculatus upon lactation performance in ewes. Journal of Agricultural Science, Cambridge. 126:353-362 and Waghorn, G. C, Shelton, I. D., McNabb, W. C ₁ and McCutcheon, S.N. 1994. Effects of condensed tannins in Lotus peduncalatus on its nutritive value for sheep. 2. Nitrogenous aspects. Journal of Agricultural Science. 123:109-119.

Tannins increase animal production in spite of reductions in the digestibility of nitrogen (i.e. more nitrogen ends up in the dung). This could be argued as a benefit of tannins, as more nitrogen goes into faeces relative to urine.

However, plants containing condensed tannins can be difficult to grow, their yield per hectare is generally lower than other grasses and legumes, and have low resistance to grazing. Therefore, their use is limited in pastoral systems.

Although some of the methods of animal production discussed, result in conversion of feeds into valuable products such as meat, milk, eggs and wool, it may also create residues which are undesirable.

None of the prior art methods mentioned above disclose a method of increasing animal production while benefiting the environment. A possible exception is condensed tannin plants which may theoretically reduce environmental nitrogen losses by reducing urinary nitrogen, although such environmental benefits are relatively untested

Therefore, it would be an advantageous to have a method of increasing animal production which is non-toxic to the animal and benefits the environment.

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 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 fi ^t s- 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 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.

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

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 INVENTION

According to one aspect of the present invention there is provided a method of increasing animal production,

characterised by the step of,

introducing a nitrification inhibitor into the digestive system of an animal.

The term 'animal production' should be taken to mean any product(s) or qualities of value derived from animals for market or research.

In preferred embodiments of the present invention the animal production which may be measured is live-weight gain. However, this should not be seen as a limitation on the present invention in any way. Other embodiments of animal production which may be measured include increased gains and quality in animal by products such as milk, wool and meat or any other such animal derived products.

In preferred embodiments of the present invention the animal will be a farm animal, and more prefei-ably dairy or beef cattle. Once again, this should not be seen as a limitation as it is anticipated the present invention could be used on other intensively farmed ruminants such as sheep, goats and so forth.

The term 'nitrification inhibitor' refers to any substance including at least one compound capable of directly or indirectly affecting the conversion of nitrogen containing compounds.

The term "nitrogen containing compound" should be taken to mean any compound which contains nitrogen as part of its chemical structure.

Specifically, a nitrification inhibitor is known to affect the conversion of ammonium to nitrate by inhibiting the bacterial species responsible for transformation of ammonium to nitrate in soil (Nitrosomonas spp).

It is desirable to use inhibitors that can be used in small amounts, and which are non-toxic to the animal. The amount of inhibitor administered will be dependent upon a number of factors, including the choice of inhibitor, as well as the body weight of the animal It is anticipated effective amounts of each inhibitor could be readily discernable to a skilled addressee.

A number of nitrification inhibitors are known in the art which may find use in the present invention. For example, a nitrification inhibitor suitable is dicyandiamide (DCD), which has been shown to inhibit nitrification of fertiliser nitrogen and animal urinary nitrogen when applied at relatively low rates and is non-toxic.

Another nitrification inhibitor which may find use is 3,4-dimethylpyrazole phosphate (DMPP).

However, this should not be seen as a limitation of the present invention. Any other nitrification inhibitor known in the art could conceivably be used in the invention.

The nitrification inhibitor may optionally also include one or more other beneficial compounds which function to improve or condition micro-organisms already present in the animal's digestive system or possibly the soil, allowing them to increase protein utilisation by the animal. Additionally, much of the nitrification inhibitor passes through unaltered in the animal urine and reduces environmental losses from the urine-nitrogen. Urine from animals is recognised as the main source of nitrogen loss to the environment in grazed pastures.

In preferred embodiments of the present invention the nitrogen inhibitor may be referred to simply as a 'nitrogen process modifier'.

A nitrogen inhibitor is introduced to an animal as a means of increasing animal production while being a carrier for the introduction of nitrogen process modifiers which affect the conversion of nitrogen containing components in the environment.

The nitrogen inhibitor not only increases animal production, but delivers the nitrogen process modifiers to the environment via animal waste. This means no or little delay occurs in modifying the conversion of nitrogen containing compounds.

Further, this ensures the nitrogen process modifiers are only delivered to the portion of pasture/soil on which the animal urinates, eliminating the need to apply the modifiers over a large area of pasture.

The nitrogen process modifiers (inhibitors) of the present invention may be introduced to the animal by a number of mechanisms.

In preferred embodiments the present invention inhibitors may be introduced to the animal through the digestive system of the animal using a delivery device.

When delivering the inhibitors via the digestive system, it is essential to ensure that inhibitors largely maintain their activity and are not denatured or inactivated through the passage of the animal's digestive system.

In some embodiments, the delivery device may be encapsulated to provide protection against full denaturation and subsequent absorption in the rumen or intestine.

The delivery device may be in the form of a sustained slow release device administered orally to an animal such as a ruminal bolus.

As understood by persons skilled in the art, a bolus is typically in the form of an elongate cylinder designed to slowly dissolve in the rumen of an animal.

One advantage of having sustained release devices is that the amount of modifiers released can be accurately known. This makes the treatment and analysis of the effects of the treatment of the animal much more precise than previously.

The bolus will preferably be composed of a solid matrix, coated with an impervious material having an opening through which the modifiers can be released, or a bio- erodeable coating which release the modifiers as they are exposed.

A wide variety of boluses and other delivery mechanisms are well known in the art

and it is anticipated these technologies could readily be adapted by a skilled addressee for use in the present invention.

In particular, the present invention may be used with a sustained slow release device consisting of a series of capsules within a housing which dissolve at a controlled rate in the stomach of the animal. One such example is the Captec™ bolus.

Other delivery mechanisms may also be used for the application of the inhibitor to the animal. These may include incorporating the inhibitor in animal feed, adding onto feed or into a water trough, or by other traditional delivery mechanisms such as drenches or injections.

The delivery device may optionally include other compounds beneficial to the animal being treated, or micro-organisms residing in the digestive tract of the animal in addition to the nitrification inhibitor. For example, the delivery device may also include trace elements such as zinc, copper and/or selenium.

Alternatively, the delivery device may include an anthelmintic in addition to the nitrification inhibitor.

It is anticipated that the use of nitrogen process inhibitors will be most effective for reducing environmental impacts over seasonal time periods such as from mid- autumn to late-winter, an extended period of approximately three to four months. As such it will be desirable to have a composition or delivery device that slowly released inhibitors over this time period. Weight gain can be at any time.

During the winter period, nitrogen is the most prone to loss by processes of leaching and/or denitrification/nitrous oxide emission.

However, this should not be seen as a limitation for other embodiments or in other embodiments. The inhibitors may be delivered over a shorter time period, with a

number of treatments administered to the animal as required.

A preferred embodiment of the present invention is the use of a nitrogen inhibitor (modifier) to increase animal live-weight.

Nitrogen modifiers in the rumen can alter the utilisation of nitrogen by the rumen microflora. If such changes in microbial metabolism result in an increased amount of non-ammonia nitrogen (i.e. more microbial protein and/or more undegraded dietary protein) reaching the small intestine, the overall effect would be similar to offering a forage with low protein degradability. Besides the effect on the total post-ruminal supply of protein, there would be expected a reduction in the energy expenditure associated to ammonia detoxication. The postulated extra supply of protein and energy will be used by the animal metabolic processes responsible for production (i.e. growth, milk production).

Part of the increase in nitrogen utilisation may also be due to nitrogen being slowly released from the nitrification inhibitor in the digestive system (i.e. the nitrification inhibitor acting as a source of slowly degradable nitrogen in the rumen).

There are secondary advantages of the nitrogen inhibitor which include slowing the rate of transformation of ammonium to nitrate in soil.

Inhibiting or modifying the conversion of nitrogen containing compounds allows more time for the nitrogen excreted in animal waste to be absorbed by plants, before being lost by soil processes such as leaching from the soil as nitrate. This will also reduce the level of nitrate entering ground water and affecting water quality in drinking water supplies.

It is also likely to reduce the levels of nitrous oxide released in the environment thus reducing levels of greenhouse gases emitted from intensive farming operations.

The present invention thus encompasses a range of different mechanisms by which nitrogen inhibitors could be introduced into the digestive system of animals to achieve an increase in animal production with a reduction in loss of nitrogen containing compounds into the environment.

Specific advantages of this approach include:

1. animals treated with certain dosages and types of nitrogen inhibitor retain nitrogen mirrored by increases in live-weight gain,

2. tests of hepatic and renal functions show no evidence of deleterious effect caused by the treatment with nitrogen inhibitors,

3. offering a broader feed base as the nitrogen inhibitor may be introduced into the digestive system by a number of methods e.g. bolus or water trough supply,

4. multiple animal production benefits including increased gains and/or quality to animal products such as milk, wool and meat,

5. reduction in loss of nitrogen containing compounds into the environment which includes both nitrate leaching and nitrous oxide emissions.

BEST MODES FOR CARRYING OUT THE INVENTION

As defined above, in its primary aspect, the present invention is directed to a novel method of increasing animal production by introducing to the digestion system of an animal a nitrification inhibitor that increases animal production and affects the conversion of nitrogen containing compounds in animal waste, once the waste has been excreted from the animal.

The invention is based upon the inventors' investigation into the delivery of nitrogen process modifiers, animal production and the modification of nitrogen containing

compounds in animal waste.

One preferred embodiment of the present invention consists of a sustained slow release device administered orally to grazing animals with targeted delivery to the digestive system then processed into animal waste, and urine in particular.

In preferred embodiments the sustained slow release device is in the form of a slow release bolus which is inserted into the rumen and which releases nitrogen transformation modifiers over a sustained period of up to four months. A bolus may additionally include a rumen-stable delivery system to deliver the modifiers post-ruminally.

A generic composition of the bolus is as follows,

i) a core comprising a substantially homogeneous mixture of:

a) a water insoluble physiologically acceptable binder comprising wax, fat, oil, fatty acid, fatty acid ester, fatty acid amide, fatty acid alcohol or the like organic compound having a melting point above 50°C;

b) a physiologically acceptable solubilising agent, such as polyethylene glycol stearate or the sodium salt of a long-chain fatty acid,

c) at least one nitrogen transformation modifier,

d) where required, a physiologically acceptable inert densifier of sufficient density and in sufficient quantities to give the bolus a minimum density of 1.5g/cm ³ ; and

ii) a coating of a physiologically acceptable material over substantially all of the surface of the core but leaving exposed a core portion whereby in use liquid in the rumen will dissolve said core allowing release of the nitrogen transformation modifier into the rumen.

One preferred embodiment of the bolus may include a nitrogen transformation modifier in the form of nitrification inhibitor such as DMPP or DCD with a binding agent such as glycerol monostearate.

The size of the bolus and the amount of modifier required will be dependent on a number of factors, including the identity and concentration of the inhibitor the body weight of the animal and the desired length of treatment. Such factors would be readily discernable to a skilled addressee.

EXAMPLE ONE

Sheep study on live-weight gain and DCD nitrogen inhibitor

Experimental Design

A modified cross-over design involving 12 sheep was used with 2 treatments and 2 time periods. During each period, the following treatments, with 6 replicate sheep, were infused via the rumen continuously over 5 days:

1. Control, 500 ml distilled water daily

2. DCD solution with 15 g in 500 ml water daily

All animals then had a 9 day non-treatment clearance/rest period, before treatments were reversed. A second 5-day infusion period then commenced, followed by a 9 day non-treatment clearance/rest period. The cross-over design meant that the 12 sheep received both treatments, thereby enabling within-sheep comparisons.

Animal Management and Sampling

Twelve Romney cryptorchid lambs (c. 35 kg live-weight at the beginning of the experiment) were implanted with custom made Teflon ruminal cannulae under

general anesthesia as described by Bermingham (2004). The sheep were placed in individual metabolism crates for two experimental periods. Each experimental period comprised 5 days for infusion of the treatments; and 9 days for collection of a 'wash-out' sample after infusion. They were fed 0.95 kg (dry basis) of a 70:30 mix of alfalfa/maize pellets and molassed alfalfa hay providing 15.5% crude protein in the total mix (dry matter basis). Animals had free access to water at all times during the course of the experiment.

All treatment solutions were infused using a peristaltic pump (Watson Marlow; Watson Victor Ltd., Auckland, New Zealand). Infusion lines were washed with warm de-ionised water between each infusion period.

A custom-made urine collection device (200 ml capacity) was tied around the middle of the sheep at the start of each day. When urination had occurred, samples were placed in sterile sample jars, frozen at -20 ⁰ C and retained for N process measurements and DCD analysis. After collection, the device was removed and subsequent urination was funnelled from the metabolism crate into a container with sufficient HCI (17% w/v) to maintain the pH of the urine below 2 to avoid ammonia volatilisation. At the end of each 24 hour period, urine volumes were recorded and samples were frozen and retained for analysis. Total faecal output over 24 h was collected into faecal collection bags harnessed to the animals. Faecal samples were placed in sterile sample jars, frozen at -20 ⁰ C and retained for DCD analysis. Urine and faeces over the 5 d collection period were pooled according to weight and analysed for measurements of N balance according to the methods described by Pinares-Patino et al. (2003). The N concentration of the faeces and urine samples was analysed in a Nitrogen Analyser 1500 (Carlo Erba Instruments, Milan, Italy) using an instrumental combustion method.

Fourteen days after the final infusion period, the sheep were slaughtered at the AgResearch Grasslands Animal Handling facility and various animal tissue samples were collected from each sheep, including muscle, fat, liver, kidney and wool. Sheep live-weights were measured at the start and end of the DCD infusion -..,, period.

Animal tissue samples were first homogenised in distilled water and centrifuged. The supernatant was then mixed in 20% trichloroacetic acid, before further centrifugation, solid phase extraction of the resulting supernatant and analysis for DCD by HPLC.

Statistical Analysis

Data for the nitrogen balance variables was analysed as a mixed model, with treatment being the fixed effect, with period, sheep and their interaction being the random effects. Estimates were calculated using restricted maximum likelihood method in Genstat v. 8.11 (2005). Significance of fixed effects was assessed by means of WaId test statistics compared to a chi-square distribution.

RESULTS

DCD in Urine and Faeces

In all sheep treated with DCD, the concentration of DCD in urine increased within the first day of infusion, doubled by the second day and remained at this level during the subsequent and last 2 days of the infusion period, after which it declined.

Sheep N Balance and Health

The apparent digestibility of dietary N was not affected by the DCD treatment (78.1 vs. 75.0 g/100 dietary N for the Control group, SED = 2.2).

The amount of nitrogen retained (dietary N input minus excreta N) was higher for the DCD-treated group (7.07 vs. -0.18 g N/d; SED = 2.23; P < 0.01). This effect on nitrogen retention was mirrored by the changes in live-weight measured during the experiments; animals treated with DCD gained weight (1.23 kg) while control animals lost weight (-0.85 kg) over the 5 d of the infusion period (SED = 0.67; P < 0.01).

The tests of hepatic and renal function showed no evidence of deleterious effects caused by the treatment. This was evident in blood analyses for GGT of 57.6 and 56.1 IU/L (SEM = 3.9, not significant) for Control and DCD treatments, respectively. Similarly, most of the other clinical parameters were not significantly affected by the treatments. The only exception was albumin, which was 26 and 24 (SEM = 0.8 ) g/L, for the Control and DCD treatments. However, both values were within the reference range for sheep (21-41 g/L).

DISCUSSION

Sheep treated with DCD had significantly higher live-weight gain than untreated sheep. This coincided with an increase in the amount of nitrogen retained by the

' sheep indicating grater retention and utilisation of dietary nitrogen. It is postulated that this was largely due to DCD interacting with rumen microflora resulting in less rumen degradation of feed protein and greater post-ruminal absorption of protein/amino compounds. Potentially, part of the increase in apparent nitrogen absorption (up to 25%) could have been due to DCD acting as a source of slowly- degradable nitrogen in the rumen.

Most of the DCD was recovered unaltered in the urine. An associated study with the urine applied to soil revealed that the DCD in it was highly effective in inhibiting nitrification of the urine-nitrogen over a total measurement period of 69 days

(Ledgard et al. paper in preparation). Thus, DCD administered into the rumen of

sheep over 5 days increased sheep growth and absorption of dietary nitrogen, and most of the DCD was excreted in urine resulting in inhibition of nitrification in soil thereby reducing the potential for environmental losses.

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 departing from the scope thereof as defined in the appended claims.