STATEMENT OF CORRESPONDING APPLICATIONS

This application is based on the Provisional specification filed in relation to New Zealand Patent Application No. 723926, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods of minimising environmental impacts from farmed animals. More specifically, the invention relates to various methods of identifying and selecting individual animals to minimise losses of urinary nitrogen to the environment, particularly in agriculture.

BACKGROUND ART

Minimising nitrogen output to the environment is receiving more attention in view of Kyoto Protocol obligations and environmental impacts. Urine produced from animals is a significant source of nitrogen pollution or load (hereafter referred to as 'nitrogen load' which incorporates either urine nitrogen concentration, urine volume or the product of both variables), particularly for countries with significant agricultural based economies.

This is because the nitrogen in urine may be leached into waterways and/or emitted as nitrous oxide (N2O) which is a potent greenhouse gas.

Although there is scientific dispute, references support the fact that, for a given level of daily urinary nitrogen excretion or nitrogen load, animals that excrete less urinary nitrogen load in each urine event would reduce the amount of nitrogen leached and N2O emitted. This is because urinary-nitrogen that is not utilised by plants or immobilised into soil organic matter is vulnerable to being lost, which increases exponentially with the nitrogen load. In New Zealand, animal urine is understood to be the primary source of nitrogen loss from pastoral agriculture, which can account for up to 90% of the country's nitrate leaching and/or urine 2O emissions from urine are also estimated to account for around 17% of NZ's greenhouse gas inventory ¹ .

Dung, by contrast, accounts for very little of these emissions. On a per hectare basis, cattle consuming the same amount of nitrogen as sheep or deer leach twice the amount of nitrogen to groundwater ² - In New Zealand, bovine animal species (such as cows) contribute the most to these statistics, however sheep, deer, pig, goat and other farmed animal species also contribute to nitrogen losses from grazed pastures.

The cost of nitrogen emissions is borne by countries and individuals. At a national level, Governments may face financial penalties from 2013 under the Kyoto Protocol greenhouse gas agreement for not meeting agreed targets for lowered emissions. At a local level, nitrogen leaching costs are already being passed on to farmers and food producers.

By way of example, a nitrogen discharge allowance (NDA) has been set for the Lake Taupo catchment in the North Island of New Zealand. An effect of this discharge allowance is that farm stock loading in the catchment area is effectively capped at a grazing intensity that does not allow the discharge allowance to be exceeded, thereby preventing further productivity growth in the region. It is anticipated that over time further discharge allowances will be in place in other areas around New Zealand and elsewhere in the world.

Methods of minimising the effect of nitrogen emissions to the environment from animals are either anthropogenic, by dealing with the nitrogen problem after the animal has excreted, or by attempting to address the problem by manipulating animal diet.

¹ de Klein & Ledgard, 2005. Nitrous Oxide Emissions from New Zealand Agriculture - key sources and mitigation strategies. Nutrient Cycling in Agroecosystems 72: 77-85

² Hoogendoorn et al. July 2008. International Grassland Congress, Hohhot, China For example, an article published in June 2005 ³ describes various nitrification inhibitor compounds can be used to slow down the rate at which ammonium from urine and faeces is converted into nitrate in the soil. The aim of such soil process inhibitors is to retain more nitrogen in the soil for plant use and thereby reduce nitrogen leaching and N2O emissions. Whilst providing a useful tool to manage nitrogen output, soil nitrification inhibitors add to the cost of farming and hence use of the inhibitors is driven primarily by legislative and market requirements. Furthermore, even nitrification inhibitors may be overwhelmed with the load of nitrogen to be handled as animals seldom graze completely randomly resulting in areas of highly concentrated excreta. Also, N ₂ 0 emissions from at least urinary nitrogen may double with soil compaction by animals where they congregate.

An example of the dietary approach is discussed in a paper written by Ishler ⁴ . In this paper, various feed regimes are described with the aim of minimising nitrogen release from animals by using various feeds and feeding regimes. It is acknowledged that this method may well work but as may be appreciated, placing animals on strict dietary regimes can be difficult or unfavourable in outdoor pasture based grazing systems.

As an example, many countries graze animals outdoors on a pasture diet and as a result it would be particularly difficult to manage the animal feed as suggested in Ishler. Even in countries which practice grain feed regimes for farmed animals, there still needs to be a balance between costs of production (including feed cost) versus that which the consumer will pay for the end product. Incorporating particular dietary regimes may reduce nitrogen output but also increase production cost to an undesirable level.

³ Ledgard, 2005. Nitrous Oxide Emissions from New Zealand Agriculture - key sources and mitigation strategies. Nutrient Cycling in Agroecosystems 72: 77- 85

⁴ http://das.psu.edu/dairy/pdf-dairy/nitrogenandammonia.pdf While the total daily nitrogen excreted in urine from each animal might be the same, there would be an advantage to the environment if that same nitrogen load were spread more evenly around the animal's area of allowable movement. This is because nitrogen leaching to groundwater increases exponentially as the load of nitrogen in a urine patch in the soil increases ⁵ .

The inventors have found that the nitrogen load per urination event, calculated from measurements of nitrogen concentration multiplied by volume, is not determined by feed or environment alone. The inventors have found that there is a degree of animal specific variation. The inventors consider that one reason for this animal specific variation may be due to genetic factors.

Tests completed by the inventors on cattle eating similar diets show a natural range of variation in urinary nitrogen concentration, urine volume and urination frequency ⁶ .

It should be appreciated that it would be useful to have a method of reducing nitrogen load that was not dependent on anthropogenic interaction and/or that is not dependent on varying animal dietary inputs.

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 that may be 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.

⁵ Ledgard et al 2015. Salt as a mitigation option for decreasing nitrogen leaching losses from grazed pastures. J. Science of Food and Agriculture 95: 3033-3040.

⁶ Betteridge & Andrewes 1986 J. Agricultural Science (Cambridge) 106:396-404. 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.

Throughout this specification, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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 a first aspect of the present invention there is provided a method of minimising the nitrogen load from animals by the steps of:

(a) testing animals to identify individual nitrogen load; and,

(b) manipulating animal gene or genes in order to reduce the average nitrogen load. In one exemplary embodiment, manipulation may be indirect via selecting animals with a low average nitrogen load for breeding using selective breeding techniques.

In an alternative exemplary embodiment, manipulation may be direct selecting animals with a low average nitrogen load for breeding using via genetic engineering techniques.

In a further exemplary embodiment, both selective breeding and genetic engineering techniques may be used.

According to a further aspect of the present invention there is provided a method of minimising the nitrogen load from animals by the steps of:

(a) testing animals for individual nitrogen load; and, (b) indirectly manipulating the animals by selectively breeding the animals that produce the lowest nitrogen load.

According to a further aspect of the present invention there is provided a method of minimising the nitrogen load from animals by the steps of:

(a) testing animals for individual nitrogen load production; and,

(b) directly manipulating the animals by genetically engineering animals in order to produce a lower nitrogen load from the animals.

The invention stems from the discovery by the inventors that the nitrogen load in a urine patch, as determined by measuring the concentration of nitrogen and/or volume of urine excreted from an animal over a period of time or within a single urination event, is not determined by feed or environment alone. That is, the inventors have found that there is a degree of animal specific variation that appears to influence the urinary nitrogen distribution within the animal's area of allowable movement. A reason for this is understood by the inventors to be related to genetic factors. The invention lies in the use of that information in various methods of animal management including the selection of animals identified as having a low nitrogen load for breeding purposes.

For the purposes of this specification, the term 'minimising' or grammatical variations thereof encompasses related verbs and phrases such as 'reduce', 'cut down', 'attenuate', 'lessen', or 'shrink' and refers to reducing nitrogen output to the environment.

The term 'nitrogen load' or grammatical variations thereof refers to the rate or amount of nitrogen per urine patch, i.e. the concentration of nitrogen in urine, the volume of urine and/or the product of combinations of these two variables over a period of time from an animal. The nitrogen load may be expressed as an average derived from the total number of urination events over a period of 24 hours (or longer) or as a single urination event. The term 'urine patch' should be understood to mean the area of ground onto which the animal discharges its urine.

It should be appreciated that an animal having a low (or lower than average) nitrogen load per urination event may still excrete a similar or even greater amount of nitrogen in urine on a daily basis than an animal with a higher nitrogen load per urination event. However, the animal will move about so each urination event may occur on a different urine patch.

The term 'genetic engineering' or grammatical variations thereof refers to direct manipulation techniques used to achieve a desired effect from an organism.

An example of genetic engineering may be to isolate genes associated with low nitrogen load from selected animals, insertion of the gene or genes into a vector, transfer of the vector to animals with higher nitrogen load and subsequent transformation. Persons skilled in the art of genetic engineering will readily appreciate how the identification, isolation and subsequent manipulation of the relevant genes will be achieved.

As should be appreciated from the above, minimising animal urinary nitrogen load in each urine patch in turn minimises release of nitrogen into the wider environment in which the animals may be grazed.

This is because each urine patch is less likely to exceed the local saturation threshold of the urine-affected area, above which exponentially greater emissions will occur. The result of such greater emissions may be leaching or runoff of nitrogen into waterways. This could be detrimental to the health of the ecosystem of the waterway.

In one exemplary embodiment, the animal urine is tested to determine the nitrogen load from the animal tested. This is achieved through measuring the total volume of the urine excreted in a single urination event and analysing its nitrogen content. For greater accuracy, this may be repeated with a pre-determined time period to calculate at an average nitrogen load per urination event.

In an alternative embodiment, other animal parameter or parameters may be measured which correlate with the nitrogen load resulting from each urination event. Parameters that may be used include a marker or markers in the milk, blood tissue or DNA from the animal.

Parameters may be tested using a non-contact sensing device, such as a video capture device, or manual recording methods, which estimate frequency and/or duration of urination, and therefore, distribution of urinary nitrogen deposition within the animal's area of allowable movement. In one exemplary embodiment, the sensing device may be an in-line sensor. In- line sensors have benefits as they avoid the need for regular batch testing and, particularly for milk, are a known and used sensing device e.g. for mastitis analysis. An alternative is an in-line sensor that measures the urine nitrogen concentration per urination event where the sensor is attached to and carried by the animal.

A further alternative is a sensor that measures the time and/or duration of each urination event that correlates with the nitrogen load resulting from each urination event. The sensor is either attached to and carried by the animal or is based on video recordings or manual observations of animals.

In one further exemplary embodiment, selective breeding noted above may be completed by the steps of: (a) testing the nitrogen load produced by a representative number of urination events from animals in similar environments and with similar diets to determine a nitrogen load per urination event; and,

(b) selectively removing animals with the highest nitrogen load per urination event over a predetermined time period. In the above exemplary embodiment, the predetermined time period may be 24 hours.

In the above exemplary embodiment, removed animals are preferably culled.

Alternatively, removed animals are relocated to a land area having a higher or no nitrogen discharge allowance. In this alternative embodiment, different geographic areas with lower risks of nitrogen emission to the environment may be used to reduce the net load on the environment through leaching or nitrous oxide emissions.

This may be preferable for animals that are otherwise of high productivity, for example, good milk producers. However, it will be appreciated that care will be required when breeding from such animals to ensure that their progeny, which may be genetically predisposed to a high nitrogen load per urination event, are not subsequently moved to a land area with a low nitrogen discharge allowance.

In an alternative exemplary embodiment, selective breeding may be completed by the steps of:

(a) testing the nitrogen load of urination events from animals in similar environments and with similar diets; and,

(b) breeding animals producing the lowest nitrogen load per urine event.

In a further alternative embodiment, selective breeding may be completed by the steps of:

(a) testing the nitrogen load per urination event from animals in similar environments and with similar diets; and, (b) both removing animals with the highest nitrogen load per urination event and breeding animals producing the lowest nitrogen load per urine event. In a further embodiment, the levels of urinary nitrogen in urination events and/or number of daily urination events may be predicted by single nucleotide polymorphism (SNP) from a tissue, blood or a milk or urine sample.

According to a further embodiment of the present invention there is provided a method of minimising nitrogen output from grazed land wherein the measured nitrogen load in urine produced by animals is measured on a routine basis, a database developed based on routine measurements, and wherein the database is subsequently used as a comparison model to determine the nitrogen load status of an unknown animal or animals.

The use of the database may also be advantageous in detecting any changes in measured nitrogen load for a given animal as it ages and/or as it changes live weight. This allows appropriate decisions regarding the management of that animal should its nitrogen load change over time.

According to a further embodiment of the present invention there is provided a method of minimising nitrogen output from grazed land wherein the measured nitrogen load in urine produced by animals is estimated by SNP on a routine basis, a database developed based on routine measurements, and wherein the database is subsequently used as a comparison model to determine the nitrogen load status of an unknown animal or animals.

It should be appreciated that the above model produced could be based on various set variables including but not limited to animal species, animal breed, animal feeds, breeding worth trait values, animal environments and so on. By having a known standard from a model, predictions may then be made for unknown individual animals or even groups of animals and the results then used in making various animal management decisions such as culling, breeding, sire selection, DNA manipulation and stock movement. Preferably, in the above methods, the animal is a non-human mammal. Many farmed animals may be used in the above methods without departing from the scope of the invention with examples including: cattle, dairy cows, sheep, deer, goats, horses, pigs, llamas and so on.

In one exemplary embodiment, the animal is a bovine species animal. In this particular embodiment, the bovine species animals may be dairy cows or beef cattle.

In an alternative exemplary embodiment, the animal is an ovine species animal. In this particular embodiment, the ovine species animals may be sheep.

In a further alternative exemplary embodiment, the animal may be a cervus species animal. In this particular embodiment, the cervus species animals may be deer. The above animals are preferred in view of their abundance in agriculture and animal husbandry and therefore these animals represent the greatest impact in minimising nitrogen emissions.

According to a further aspect of the present invention there is provided a method of minimising the amount of nitrogen leached from grazed land by manipulating the genes of animals that graze the land in order to produce the lowest nitrogen load in the animal's individual urine events.

According to a further aspect of the present invention there is provided a method of reducing N ₂ 0 emissions from grazed land by manipulating the genes of animals that graze the land in order to produce the lowest nitrogen load in the animal's urine. According to a further aspect of the present invention there is provided a method of animal management to reduce the level of nitrogen emitted to the environment by use of the method substantially as described above.

In one exemplary embodiment, manipulation described in the above methods is indirect via selective breeding techniques. In an alternative exemplary embodiment, manipulation described in the above methods is direct via genetic engineering techniques.

In a further exemplary embodiment, both selective breeding and genetic engineering techniques may be used. According to a further aspect of the present invention, there is provided the use of a sensor device to test animal nitrogen load in the animal's urination events, the number of daily urination events and combinations thereof, as a means to minimise the impact of nitrogen emitted to the environment from the tested animal's urine, wherein nitrogen emitted is measured in terms of nitrogen leaching; N ₂ 0 emissions and combinations thereof. It should be appreciated from the above description that there are described various methods of minimising nitrogen load from an animal's individual urination events. The methods utilise the discovery that animals naturally produce varying nitrogen loads in urination events and manipulating the animal genes by selectively breeding animals or genetically engineering animals are techniques that may be used to minimise nitrogen emissions to the environment. Advantages of the methods should be apparent to those skilled in the art include the fact that the method is relatively simple, requires minimal expense on the part of herd management and deals with the problem at the source rather than other methods such as nitrification inhibitors which deal with emissions post deposition.

More specifically, the use of nitrogen inhibitors or dietary modifications incurs ongoing costs associated with materials or labour input throughout the life of each animal, whereas the use of selective breeding and/or genetic engineering does not suffer from these disadvantages.

Over time, selective breeding of animals with relatively low nitrogen loads will arrive at entire herds genetically predisposed to this characteristic with the potential benefit of reduced nitrogen being emitted into the environment. This has favourable implications for the health of the ecosystem.

BRIEF DESCRIPTION OF THE 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 is a graph showing measured u rine volume and the number of urination events per day for two individual steers;

Figure 2 is a graph showing measured urine volume for a group of cows fed simple pasture and a group of cows fed diverse pasture;

Figure 3 is a graph showing measured urine load for a group of cows fed simple pasture and a group of cows fed diverse pasture;

Figure 4 is a graph demonstrating the relationship between the u rination duration and urine nitrogen load.for a cow fed simple pasture; and

Figure 5 is a graph demonstrating the relationship between the time between urination events and urine nitrogen load for a cow fed simple pasture.

BEST MODES FOR CARRYING OUT THE INVENTION

The invention is now described with reference to a trial completed by the inventors to determine whether variation in nitrogen loads and volumes in individual animal urination events have an animal specific variation not influenced by animal diet or environment.

EXAMPLE 1 Two steers were selected and a custom-made device attached to each one which monitored the geographical position, start time, duration, nitrogen concentration and volume of each urination event.

On-animal devices allowed the steers to move freely about and behave naturally in their environment, as opposed to a technique involving manually gathered samples which would not have provided this advantage.

During device validation, a sample of the urine was collected and stored for later laboratory analysis. The laboratory analysis had a high precision and accuracy for the purposes of validation of the on-animal sensors. The on-animal devices were found to have sufficient accuracy and precision. All steers were fed the same diet and exposed to the same environmental conditions.

The resulting measured urine volume, and number of urination events per day, varied greatly between the individual steers (Figure 1).

Subsequent research by the inventors has shown significant variation in urinary nitrogen concentration within and amongst animals in a species, when grazed in a common environment as shall be seen in the following example.

EXAMPLE 2

A total of twenty lactating dairy cows were allocated to two treatment groups (10 cows per group) that grazed either Simple (standard ryegrass-white clover pasture) or Diverse pasture (mixture of grass, herbs and legumes). Cow live-weight was balanced across the treatments.

Each cow was fitted with a custom-made urine sensor device that measured the time, duration, volume (litres) and nitrogen (%N) concentration of each urination event over a 96 hour period. The nitrogen load (grams of nitrogen per urination event) was calculated using the volume and nitrogen concentration of each urination event. The results from the study showed that the volume of each urination event averaged 2.35 litres across cows with no significant effect of pasture treatment on urine volume. There was statistically significant (P < 0.01) variation between individual cows in urine volume with a standard deviation of 0.37 litres (Figure 2). The nitrogen load averaged 14.6 g nitrogen per urine event across cows with no significant effect of pasture treatment on nitrogen load per urination event. There was statistically significant (P < 0.01) variation between individual cows in the nitrogen load per urination event with a standard deviation of 3.8 g nitrogen per urination event (Figure 3).

There is a strong relationship between the urination duration and urine nitrogen (N) load measured over the trial (Pearson correlation coefficient of R = 0.54; with a statistical significance of P = 0.001) (cow 177, Simple treatment group) (Figure 4). The contour lines represent the multivariate lognormal distribution fit to the data.

There is also a very strong relationship between the time between urination events and urine nitrogen (N) load measured over the trial (Pearson correlation coefficient of R = 0.89; with a statistical significance of P = 0.001) (cow 177, Simple treatment group) (Figure 5). The contour lines represent the multivariate lognormal distribution fit to the data.

EXAMPLE 3

A total of thirty-two lactating dairy cows were allocated to four treatment groups (10 cows per group) that grazed pasture (standard ryegrass-white clover pasture) under Current or Future management systems in December and April. Cow live-weight was balanced across the treatments.

Each cow was fitted with a custom-made urine sensor device that measured the time, duration, volume (litres) and nitrogen (%N) concentration of each urination event over a 96 hour period. The nitrogen load (grams of nitrogen per urine per urination event) was calculated using the volume and nitrogen concentration of each urination event.

The average urine nitrogen concentration was 0.55 %. There was a statistically significant between-cow variance in the urine nitrogen concentration in April and December, with a standard deviation of 0.086 % nitrogen per urination event in December (P < 0.01) and a standard deviation of 0.071 % nitrogen per urination event in April (P < 0.05).

The average nitrogen concentration per urination event was also repeatable between April and December (Pearson correlation coefficient of R = 0.96; P < 0.01); i.e. cows with low or high average nitrogen concentrations in April, also had low or high average nitrogen respective concentrations in December. ⁷ This demonstrates that the between cow variability in urine parameters are repeatable between seasons.

These results indicate that by careful and selective breeding of cows with lower nitrogen loads per urine event, and/or greater number of urine events per day, that the total nitrogen emissions leached by such a herd may be reduced. Alternatively, cows producing higher loads of nitrogen in urine patches may be culled or both culling and breeding techniques used in order to minimise the nitrogen emissions to the environment.

Given similarities between animals it is a reasonable expectation that other animals such as sheep, goats, pigs, deer etc. will exhibit similar variation and hence similar herd management techniques may be employed. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

⁷ Shepherd et al., 2017. Evaluation of urine excretion from dairy cows under two farm systems using urine sensors. Agriculture, Ecosystems and Environment 236: 285-294 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.