TECHNICAL FIELD

The present invention is generally concerned with improved birth rates in mammals. The present invention provides methods and compositions for increasing the probability of a live birth in a gestational mammal, for example a bovine animal or a herd of bovine animals, which methods and compositions comprise the pre-treatment of gametes or embryos with an allene oxide synthase prior to their use in an assisted reproduction technique.

BACKGROUND OF THE INVENTION

Allene oxide synthase (AOS) is a cytochrome p450 enzyme family member (CYP74A; EC 4.2.1.92) first isolated from the guayule rubber plant Parthenium argentatum (GenBank CAA55025.2). Also known as the guayule rubber particle protein (RPP), AOS has been purified and cloned from the guayule rubber plant (US 5,633,433 and US 6,132,711).

AOS is an antioxidant enzyme with specificity for lipid peroxides in biological systems. As reported in US 6,132,711, AOS rapidly converts free or esterified fatty acid peroxides or hydroperoxides into their corresponding epoxides which are, in turn, converted to ketols. The lipid peroxide and hydroperoxide substrates for this enzyme are said to be toxic to biological organisms and can generate additional peroxides by chain propagation reactions as well as causing oxidative damage to proteins and DNA.

The accumulation of lipid peroxidation products has been identified as a major contributor to cell damage, dysfunction, and death. These products have been linked to the pathology of several ageing and oxidative stress related diseases, and therefore represent a major target for biotechnology applications. This topic is an emerging research field, and functional roles for lipid peroxidation products in disease states are now being identified. Consequences include altered gene regulation, signalling and receptor activation. AOS enzymes convert polyunsaturated fatty acid (PUFA) oxidative derivatives to the corresponding epoxide through stereospecific dehydration by intramolecular oxygen transfer as part of the jasmonic acid biosynthesis pathway in plants. It is reasonable to suggest that outside of plants, AOS enzymes could act as antioxidant molecules to scavenge peroxidation products and prevent chain oxidation reactions, making them commercially valuable for several applications. Applicants' current allene oxide synthase (AOS) enzyme from Parthenium argentatum (trade name "Aloxsyn") shows excellent activity against linoleic hydroperoxide, the peroxidation product of linoleic acid, an essential fatty acid readily found in biological systems.

Applicants previous work focused on the antioxidant effects of allene oxide synthase on sperm and embryos in the contest of enhancing the longevity and/or viability of fertilised/gametes. This led to the hypothesis that allene oxide synthase treated gametes or embryos may lead to enhanced animal conception or pregnancy rates when used in artificial or assisted reproduction practices.

Indeed a large scale, statistically powered, trial was recently conducted on a commercial dairy farm to determine effects of semen treatment with Aloxsyn on the fertility of 3579 dairy cows following artificial insemination (AI) with the semen.

Unexpectedly, the results from this on-farm trial failed to demonstrate any positive correlation between increased pregnancy rates for cows inseminated with Aloxsyn treated sperm. In fact, the Aloxsyn treatment groups performed worse that the non-enzyme controls.

Instead, the results of the on-farm trial surprisingly demonstrated that animals artificially inseminated with Aloxsyn treated sperm yielded a higher overall proportion in live births of viable calves (also referred to herein as "calving rate") when compared to control groups artificially inseminated with untreated sperm.

Accordingly, the present invention provides novel methods and compositions which improve the probability of live births in gestational mammals, including bovine animals. In particular, the methods and compositions described herein are likely to have a significant social and/or economic impact on existing breeding and farming practices, particularly in the dairy and beef farming industries where milk and meat production is often in high demand.

SUMMARY OF THE INVENTION

The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary of the Invention. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.

In an aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the gestational mammal comprising the AOS contacted gamete thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal not used in an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a cattle animal, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the cattle animal comprising the AOS contacted gamete thereby increasing the probability of a live birth in the cattle animal relative to a cattle animal not subjected to an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a bovine animal, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the bovine animal comprising the AOS contacted gamete thereby increasing the probability of a live birth in the bovine animal relative to a bovine animal not subjected to an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a human, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the human comprising the AOS contacted gamete thereby increasing the probability of a live birth in the human relative to a human who did not undergo an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a herd of cattle animals or a herd of bovine animals, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on at least one animal in the herd of cattle animals or at least of one animal in the herd of bovine animals comprising the AOS contacted gamete thereby increasing the probability of a live birth in the herd of cattle animals or the herd of bovine animals relative to a herd of cattle animals or herd of bovine animals not subjected to an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a gamete for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the gamete has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a use of a gamete for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the gamete has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a veterinary composition for increasing the probability of a live birth in an assisted reproduction technique in a gestational mammal, the veterinary composition comprising a gamete and an allene oxide synthase. In a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to the gestational mammal in an assisted reproduction technique thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal who did not receive an AOS contacted embryo transfer.

In a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a cattle animal, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to the cattle animal in an assisted reproduction technique thereby increasing the probability of a live birth in the cattle animal relative to a cattle animal who did not receive an AOS contacted embryo transfer.

In a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a bovine animal, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to the bovine animal in an assisted reproduction technique thereby increasing the probability of a live birth in the bovine animal relative to a bovine animal who did not receive an AOS contacted embryo transfer.

In a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a human, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to the human in an assisted reproduction technique thereby increasing the probability of a live birth in the human relative to a human who did not receive an AOS contacted embryo transfer.

In a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a herd of cattle animals or a herd of bovine animals, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to at least one animal in the herd of cattle animals, or at least one animal in the herd of bovine animals, in an assisted reproduction technique thereby increasing the probability of a live birth in the herd of cattle animals or the herd of bovine animals relative to a herd of cattle animals or herd of bovine animals who did not receive an AOS contacted embryo transfer. In another aspect of the present invention there is provided an embryo for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the embryo has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a use of an embryo for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the embryo has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a veterinary composition for increasing the probability of a live birth in an assisted reproduction technique in a gestational mammal, the veterinary composition comprising an embryo and an allene oxide synthase.

In an aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the gestational mammal comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a cattle animal, the method comprising the steps of:

(i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the cattle animal comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the cattle animal relative to a cattle animal not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a bovine animal, the method comprising the steps of:

(i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the bovine animal comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the bovine animal relative to a bovine animal not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a human, the method comprising the steps of: (i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,- and

(ii) performing an assisted reproduction technique on the human comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the human relative to a human who did not undergo an assisted reproduction technique comprising the AOS contacted sperm or semen.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a herd of cattle animals or a herd of bovine animals, the method comprising the steps of:

(i) contacting sperm or semen with an allene oxide synthase (AOS) in vitro,- and

(ii) performing an assisted reproduction technique on at least one animal in the herd of cattle animals, or at least of one animal in the herd of bovine animals, comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the herd of cattle animals, or the herd of bovine animals, relative to a herd of cattle animals, or herd of bovine animals, not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In another aspect of the present invention there is provided a sperm or semen for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the sperm or semen has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a use of a sperm or semen for increasing the probability of a live birth in an assisted reproduction technique comprising a gestational mammal, wherein the sperm or semen has been contacted in vitro with an allene oxide synthase (AOS) prior to its use in the assisted reproduction technique.

In another aspect of the present invention there is provided a veterinary composition for increasing the probability of a live birth in an assisted reproduction technique in a gestational mammal, the veterinary composition comprising a sperm or semen and an allene oxide synthase.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the coding system used in the on-farm animal trial. The first three digits define the treatment group coding while the last five digits define the unique bull identifier as the source of sperm used in the trial.

Figure 2 shows the confirmed pregnancy rates for the various treatment groups used in the on-farm trial. The treatment groups 3 and 8 represent animals inseminated using non- Aloxsyn-treated sperm controls whereas treatment groups 15 and 21 represent animals inseminated with Aloxsyn-treated sperm. Figure 3 shows a bar graph depicting confirmed pregnancy rates plotted against individual bull used in the on-farm trial (i.e. 'Legendary', 'Avril', 'Transformer', 'Archilles' and 'Pango').

Figure 4 shows a line graph depicting confirmed pregnancy rates plotted against individual bull as a function of the various treatment groups used in the on-farm trial.

Figures 5A-5E show a line graph depicting confirmed pregnancy rates plotted against the various treatment groups for each individual bull.

Figure 6 shows the days from calving when bred plotted against calving date.

Figure 7 shows the days from calving when bred plotted against calving date as a function of individual treatment groups used in the on-farm trial.

Figure 8 shows the pregnancy rate plotted against treatment group for the various pregnancy rate fixed effect models.

Figure 9 shows the AOS protein sequence derived from Parthenium argentatum (GenBank: CAA55025.2; PaAOS), as defined by SEQ ID NO: 1.

Figure 10 shows a variant AOS protein sequence defined by SEQ ID NO: 2.

Figure 11 shows the AOS protein sequence derived from Solanum tuberosum (GenBank: AAN37417.1; StAOS), as defined by SEQ ID NO: 3.

Figure 12 shows the AOS protein sequence derived from Solanum lycopersicum (NCBI Reference Sequence: NP_001234833; SIAOS), as defined by SEQ ID NO: 4.

Figure 13 shows the AOS protein sequence derived from Arabidopsis thaliana (NCBI Reference Sequence: NP_199079.1; AtAOS), as defined by SEQ ID NO: 5.

Figure 14 shows the AOS protein sequence derived from Zea mays (GenBank: AAR33048.1; ZmAOS), as defined by SEQ ID NO: 6.

DEFINITIONS General Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley 8i Sons (including all updates until present).

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

Throughout this specification the word "comprise", or variations 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.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Any example described herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise. Selected Definitions

The term "about" is used herein to refer to defined values (e.g., amounts, concentrations, time etc) that vary by as much as 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to the defined value.

The term Aloxsyn as used herein refers to an enzyme preparation comprising allene oxide synthase derived from Parthenium argentatum (e.g. SEQ ID NO: 1) buffered in 0.02M Tris HCI (pH 8.2) containing 20 mM NaCI.

The term "allene oxide synthase" or "AOS" as used in this specification is intended to mean any enzyme that converts lipoxygenase-derived fatty acid hydroperoxides to allene epoxides (which are precursors of the growth regulator jasmonic acid in plants), and includes for example an allene oxide synthase isolated from the rubber plant Parthenium argentatum. These terms also include any functionally equivalent peptide or protein of an AOS, and includes AOS obtained from any source or by any method, for example by chemical synthesis and/or gene expression or cloning techniques. Any reference to AOS in this specification should be taken to include reference to functionally equivalent variants thereof, unless otherwise indicated.

The term "biologically active fragment" as used herein means a fragment of a full-length polypeptide, peptide or protein which fragment retains an activity of the polypeptide, peptide or protein. As used herein, the term "biologically active fragment" includes deletion mutants and small poly/peptides, for example of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, contiguous amino acids, which comprise an activity of the parent peptide polypeptide. Peptides of this type may be obtained through the application of standard recombinant nucleic acid techniques as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al. CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Alternatively, peptides of this type may be synthesised using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989), see particularly Chapter 9, or by Roberge et al. (1995 Science 269: 202). Alternatively, peptides can be produced by digestion of a polypeptide of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques

As used herein, "culturing", "culture" and the like refer to the set of procedures used in vitro where an embryo or population of cells (or a single cell) is incubated under conditions which have been shown to support the growth or maintenance of the cells in vitro. The art recognises a wide number of formats, media, temperature ranges, gas concentrations etc. which need to be defined in a culture system. The parameters will vary based on the format selected and the specific needs of the individual who practices the methods herein disclosed. However, it is recognised that the determination of culture parameters is routine in nature.

The term "derivative" as used herein is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent molecules.

The term "effective amount", in the context of a particular utility of the peptides and enzymes described herein, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that stimulation, prevention or treatment. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

As used in this specification, the term "fragment" or "functional derivative" in relation to a polypeptide is a subsequence of a polypeptide that may be detected (e.g.) using a binding agent. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.

The term "herd" as used herein is intended to refer to a "herd of animals". This term should be taken broadly to include any group of animals of the same species, and should also encompass other collective nouns used to refer to a group of animals of any particular species, such as a "flock" or a "drove".

The term "isolated" as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequences may be prepared by at least one purification step.

The terms "mammal" and "gestational mammal" as used herein includes, without limitation, humans, bovines (e.g. cattle), ovines (e.g. sheep), caprines (e.g. goats), porcines (e.g. pigs), equines (e.g. horses), canines (e.g. dogs), felines (e.g. cats), camelids (e.g. camels, alpacas, llamas) and marine mammals (e.g. dolphins, whales). The term "PaAOS" as used herein refers to allene oxide synthase as derived from Parthenium argentatum. An example PaAOS enzyme referred to in this specification is known as Aloxsyn (SEQ ID NO: 1).

The terms "peptide" and "polypeptide" or "protein" may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the present invention may be purified natural products, or, may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof. Polypeptides herein may have chain lengths of at least 4 amino acids, at least 5 amino acids, or at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all 23 amino acids of the full-length allene oxide synthase enzymes described herein. Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.

The term "purified" as used herein does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample. The term should be similarly understood in relation to other molecules and constructs described herein.

The terms "semen" and "sperm" are used interchangeably in this specification to refer to a sample that contains either sperm or semen (i.e.) ejaculate comprising seminal fluid and sperm. A person skilled in the art will recognise that it may be advantageous in some cases to contact a semen sample with an allene oxide synthase according to the methods described herein, or in other cases, it may be advantageous to first purify sperm from semen and then contact the purified sperm sample with an allene oxide synthase as described herein.

The term "variant" as used herein refers to polypeptide sequences different from the specifically identified sequences, wherein 1 to 6 or more or amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of one, two, three, four, five or six amino acids are contemplated. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "variant" with reference to polypeptides encompasses all forms of polypeptides as defined herein.

Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the present invention. With regard to polypeptides, identity is found over a comparison window of at least 5 to 7 amino acid positions.

Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance.

Polypeptide sequence identity and similarity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

The similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp. The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Variant polypeptide sequences commonly exhibit an E value of less than 1 x 10 ^-5 , less than 1 x 10 ^-6 , less than 1 x 10 ^-9 , less than 1 x 10 ^-12 , less than 1 x 10 ^-15 , less than 1 x 10 ^-18 or less than 1 x 10 ^-21 when compared with any one of the specifically identified sequences. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www. ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention.

The term sequence "identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lieu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

DETAILED DESCRIPTION

A large scale, statistically powered, trial was conducted on a commercial dairy farm to determine effects of allene oxide synthase (AOS) treated sperm/semen on the fertility of dairy cows following artificial insemination (AI).

The AOS used in the on-farm trial is a variant of a plant enzyme produced by fermentation using a GMP applicable biotechnology manufacturing process. The enzyme is highly purified and formulated in a buffered solution (i.e.) AOS enzyme (SEQ ID NO: 1) buffered in 0.02M Tris HCI (pH 8.2) containing 100 mM NaCI.

The anticipated outcome of the trial was to demonstrate increased pregnancy rates in AOS treatment groups, as determined by pregnancy vet inspection at >80 days gestation. The effect of AOS on calving rates was also monitored to determine effects on the quality of the pregnancy and calf health.

Allene oxide synthase was added to semen prior to freezing in the otherwise standard semen manufacturing process used by an animal genetics company partner. The AOS treated sperm/semen was included in extended semen straws prior to freeze/thaw or included in extended semen straws directly prior to artificial insemination.

Mixed model analysis was undertaken to dissect interactions between treatment and other variables including bull, cow age, days in milk (as an indicator of cycles required to achieve pregnancy), breeding technician and breeding type.

The results of the on-farm trial are summarised in Fig. 1 read in conjunction with Table 4 in Example 2. These data show that the highest pregnancy rate was achieved in a milk- based extender control (i.e. Treatment Group 3; 41.1%). This compares to 39.1% for the Optixcell Extender control (Treatment Group 8) and 38.2% for Optixcell + AOS (Treatment Group 15). Treatment Group 21, where AOS was supplemented via a second straw administered at the time of artificial insemination had the lowest pregnancy rate (36.0%). Refer to Example 1 for further details of the treatments used in the on-farm trial.

Accordingly, AOS treatment of sperm/semen failed to demonstrate an increase in overall pregnancy rates in this trial. In particular, compare Treatment Group 8 (Optixcell Extender Control; 39.1%) with Treatment Group 15 (Optixcell + AOS; 38.2%). While the highest pregnancy rate was achieved in the milk-based extender control (Treatment Group 3), the 2% difference between this and the Optixcell control group (Treatment Group 8) was not statistically significant given the numbers of animals used in the on-farm trial.

Notwithstanding the nil-effect observed for AOS treated sperm/semen on enhanced pregnancy rates, the Applicants discovered a statistically significant (3.4%) increase in the percentage of live births in an AOS treatment group (Treatment Group 15) relative to its control (Treatment Group 8). Refer to Table 6 in Example 3. This result was both surprising and unexpected.

Accordingly, in an aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the gestational mammal comprising the AOS contacted gamete thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal not subjected to an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the gestational mammal comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In yet another aspect of the present invention there is provided a method for increasing the probability of a live birth in a gestational mammal, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to the gestational mammal in an assisted reproduction technique thereby increasing the probability of a live birth in the gestational mammal relative to a gestational mammal who did not receive an AOS contacted embryo transfer.

In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 1.

In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 2. In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 3.

In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 4.

In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 5.

In an example according to these and other aspects of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 6.

In a further example according to these and other aspects of the present invention, the allene oxide synthase comprises an allene oxide synthase in a pH buffered solution.

In yet a further example according to these and other aspects of the present invention, the concentration of allene oxide synthase used is between 0.1 and 100 ug/mL. For any avoidance of doubt, a concentration of allene oxide synthase between 0.1 and 100 ug/mL includes, without limitation, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,

1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,

3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4,

5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,

7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,

9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 367, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 525, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99 and 100 ug/mL allene oxide synthase.

In a further example according to these and other aspects of the present invention, the assisted reproduction technique is selected from artificial insemination (AI), in vitro fertilisation (IVF) including embryo implant, intracytoplasmic sperm injection (ICSI), gamete intrafallopian transfer (GIFT), and the like.

In yet a further example according to these and other aspects described herein, the step of contacting the gamete or embryo with an allene oxide synthase comprises admixing the AOS with the gamete or embryo for a time and under conditions necessary to increase the probability of a live birth in the gestational animal.

In yet a further example according to these and other aspects described herein, the step of contacting the gamete or embryo with an allene oxide synthase comprises culturing the gamete or embryo with the AOS for a time and under conditions necessary to increase the probability of a live birth in the gestational animal.

In yet another example according to these and other aspects of the present invention, the gestational mammal includes, but is not limited to, a cattle animal and a human. In a related example, the cattle animal is selected from the Bovidae family, Equidae family, Camelidae family and the Suidae family.

Examples of animals from the Bovidae family include the Bovinae subfamily. Animals from the Bovinae subfamily include, but are not limited to, Bos taurus and Bos indicus. This comprises beef and/or dairy breeds such as Jersey, Holstein, Friesian, Ayrshire, Angus, Hereford, Simmental, Romosinuano, Criollo, Carora, Senepol as well as other crossbreds.

Examples of animals from the Bovidae family also include the Caprinae subfamily. Examples of animals from the Caprinae subfamily include, but are not limited to, Capra hicus. This comprises breeds such as Saanen, Alpine, Nubian, Boer and Cashmere.

Further examples of animals from the Caprinae subfamily include the Ovis genus. This comprises breeds such as Border Leicester, Merino, Wiltshire, Dorset, East Friesian, and Lacaune.

Examples of animals from the Equidae family include the Equus subfamily. Animals from the Equus subfamily include, but are not limited to, Equus ferus caballus. This comprises breeds such as

Examples of animals from the Camelidae family include theLamini and Camelini subfamily. This includes animals such as camels, alpacas and llamas.

Examples of animals from the Suidae family include the Suinae subfamily. Animals from the Suinae subfamily include, but are not limited to, the Sus genus. This comprises breeds such as Landrace, Duroc, Meisham, Berkshire and Philippine native.

The methods of the present invention are particularly useful in a beef and milk farming context where it is desirable to enhance the number of live births achieved in any given breeding cycle. In turn, this increases animal numbers available for milking or for meat production.

Accordingly in a further aspect of the present invention there is provided a method for increasing the probability of a live birth in a Bovidae animal, the method comprising the steps of:

(i) contacting a gamete with an allene oxide synthase (AOS) in vitro,· and

(ii) performing an assisted reproduction technique on the Bovidae animal comprising the AOS contacted gamete thereby increasing the probability of a live birth in the Bovidae animal relative to a Bovidae animal not subjected to an assisted reproduction technique comprising the AOS contacted gamete.

In another aspect of the present invention there is provided a method for increasing the probability of a live birth in a Bovidae animal, the method comprising the steps of:

(i) contacting a sperm or semen with an allene oxide synthase (AOS) in vitro,· and (ii) performing an assisted reproduction technique on the Bovidae animal comprising the AOS contacted sperm or semen thereby increasing the probability of a live birth in the Bovidae animal relative to a Bovidae animal not subjected to an assisted reproduction technique comprising the AOS contacted sperm or semen.

In yet another aspect of the present invention there is provided a method for increasing the probability of a live birth in a Bovidae animal, the method comprising the steps of:

(i) contacting an embryo with an allene oxide synthase (AOS) in vitro,·

(ii) transferring the AOS contacted embryo to a surrogate Bovidae animal in an assisted reproduction technique thereby increasing the probability of a live birth in the Bovidae animal relative to a Bovidae animal who did not receive an AOS contacted embryo transfer.

In an example according to these and other aspects of the present invention, the Bovidae animal is Bos taurus or Bos indicus.

The present invention further contemplates products for use in an artificial reproduction technique to increase the number of live births in a gestational animal.

Accordingly, in another aspect of the present invention there is provided a veterinary composition for use in increasing the probability of a live birth in an assisted reproduction technique in a gestational mammal, the veterinary composition comprising a gamete and an allene oxide synthase.

In an example according to these and other aspects of the present invention, the gamete is a sperm or an oocyte.

Accordingly, in another aspect of the present invention there is provided a veterinary composition for use in increasing the probability of a live birth in an assisted reproduction technique in a gestational mammal, the veterinary composition comprising a sperm or semen and an allene oxide synthase.

In yet another aspect of the present invention there is provided a veterinary composition for use in increasing the probability of a live birth in an assisted reproduction technique involving a gestational mammal, the veterinary composition comprising a sperm, semen or an embryo together with an allene oxide synthase.

In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 1.

In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 2. In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 3.

In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 4.

In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 5.

In an example according to the veterinary compositions of the present invention, the allene oxide synthase comprises or consists in an allene oxide synthase defined by SEQ ID NO: 6.

In a further example according to the veterinary compositions of the present invention, the allene oxide synthase comprises an allene oxide synthase in a pH buffered solution.

In yet a further example according to the veterinary compositions of the present invention, the concentration of allene oxide synthase used is between 0.1 and 100 ug/mL. For any avoidance of doubt, a concentration of allene oxide synthase between 0.1 and 100 ug/mL includes, without limitation, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,

1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,

5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,

7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,

9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 367, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 48, 49, 50, 51, 525, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70

71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

95, 96, 97, 98, 99 and 100 ug/mL allene oxide synthase.

In yet another example according to the veterinary compositions of the present invention, the composition further comprises an extender to extend the life of the gamete or embryo for use in the assisted reproduction technique.

Examples of extenders according to the present invention includes, but is not limited to, Optixcell (IMV technologies, Ref. 026218-025239), Triladyl (Minitube, Ref. 13500/0250) and Andromed (Minitube, Ref. 13503/0200) AOS Enzymes

The methods and/or compositions of the present invention are directed to an allene oxide synthase which comprises, consists or consists essentially of an amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 6. By definition, this includes variants or fragments of any one of SEQ ID NOs: 1 to 6, provided that the variant enzymes or fragments retain allene oxide synthase activity.

As used herein, the term "comprises", "comprising" and the like means the enzyme includes the amino acid sequence corresponding to a sequence defined by any one of SEQ ID NOs: 1 to 6, and may also include any one or more other elements. Thus, the amino acid sequence corresponding to any one of SEQ ID NOs: 1 to 6 is a mandatory element, and any other elements are optional and may or may not be present. Other elements may include, for example, additional amino acid residues at either end of the amino acid sequence, and/or other molecules.

As used herein, the term "consists essentially of" (and the like) means that the allene oxide synthase enzyme includes the amino acid sequence corresponding to a sequence defined by any one of SEQ ID NOs: 1 to 6, and may also include one or more other elements, provided those elements do not interfere with or contribute to the activity or action of the peptide. Thus, the amino acid sequence corresponding to a sequence defined by any one of SEQ ID NOs: 1 to 6 is a mandatory element, and other elements are optional and may or may not be present, depending upon whether or not they affect the activity or action of the enzyme. For example, where a composition "consists essentially of an amino acid sequence corresponding to the sequence SEQ ID NO: l", the amino acid sequence may comprise additional amino acid residues (e.g., by as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more additional residues) at either end of the amino acid sequence, and/or may be conjugated or otherwise associated with other molecules (e.g., a protecting moiety such as an N-terminal blocking residue (e.g., pyroglutamate)), provided those additional residues or molecules do not substantially modulate the enzymatic properties of the amino acid sequence.

As used in the above statement and in similar statements elsewhere in this specification, the term "consists of" (and the like) means that the peptide includes, and is limited to, the amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6. Thus, the phrase "consists of" indicates that the amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 is a mandatory element, and that no other elements (such as amino acid residues at either end of the amino acid sequence or other molecules) may be present.

As used in the statement "amino acid sequence corresponding to the sequence SEQ ID NO: l" and similar statements in this specification, the term "corresponding to" or "corresponds to" (and the like) means an amino acid sequence that displays substantial similarity and/or identity to the sequence defined by any one of SEQ ID NOs: 1 to 6, and that possesses the desired enzymatic activity. In general, allene oxide synthase employed in the methods and compositions described herein displays at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 % similarity and/or identity to the sequence defined by any one of SEQ ID NOs: 1 to 6.

In certain examples, the sequence of the amino acid may differ from any one of SEQ ID NOs: 1 to 6 by at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 amino acid substitutions, additions, and/or deletions.

Substituted amino acids may include conservative amino acid substitutions, as well as non-conservative substitutions.

The term "conservative amino acid substitutions" as used in this specification is intended to mean the substitution of amino acids that have similar biochemical properties. It will be appreciated that appropriate conservative amino acid substitutions are based on the relative similarity between different amino acids, including the similarity of the amino acid side chain substituents (for example their size, charge, hydrophilicity, hydrophobicity and the like). By way of example, a conservative substitution includes substitution of one aliphatic amino acid for another aliphatic amino acid, substitution of an amino acid having an hydroxyl- or sulphur-containing side chain with another amino acid having an hydroxyl- or sulphur- containing side chain, substitution of an aromatic amino acid with another aromatic amino acid, substitution of a basic amino acid with another basic amino acid, or substitution of an acidic amino acid with another acid amino acid. Examples of conservative amino acid substitutions include: substitution of glycine, alanine, valine, leucine, or isoleucine, one for another substitution of serine, cysteine, threonine, or methionine, one for another substitution of phenylalanine, tyrosine, or tryptophan, one or another substitution of histidine, lysine, or arginine, one for another substitution of aspartic acid, glutamic acid, asparagine or glutamine, one for another Alternatively, or in addition, substituted amino acids or added amino acids can be any non-naturally occurring amino acids or derivatives thereof. Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of non-naturally occurring amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.

In some examples, the allene oxide synthase enzyme that comprises, consists or consists essentially of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 is or is a derivative of a homolog or isoform of the sequence defined by any one of SEQ ID NOs: 1 to 6, or displays at least about 80, 81, 82, 83, 84, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % similarity and/or identity to a homolog or isoform of the sequence defined by any one of SEQ ID NOs: 1 to 6. A "homolog" is a molecule from a different species and which is related by descent from a common ancestral DNA sequence. The term "homolog" may apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication. An "isoform" is a peptide or enzyme that has the same function as another peptide or enzyme but which is encoded by a different polynucleotide and may have small differences in its sequence.

The allene oxide synthase enzyme comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 may be a biologically active fragment of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6. Reference herein to a "fragment" means a molecule which contains at least about 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 or about 480 contiguous amino acids. The biologically active fragment may have the activity associated with the full-length amino acid sequence and/or may have an altered activity. An "altered activity" includes an enhanced activity or loss of a detrimental activity. The biologically active fragment may have the ability to convert free or esterified fatty acid peroxides or hydroperoxides into their corresponding epoxides, as described herein.

Methods well known in the art can be used to determine whether an amino acid sequence is a peptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 as herein defined.

The polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 may comprise a peptide that has been suitably modified, for example, by lipid modification to modify its physico-chemical properties.

The polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by any one of SEQ ID NOs: 1 to 6 may be prepared in recombinant form using standard protocols as, for example, described in Sambrook et al. MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbour Press, 1989), in particular Sections 16 and 17; Ausubel et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Typically, the peptide may be prepared by a procedure including the steps of (a) providing an expression vector from which the peptide is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant peptide from the vector; and (d) isolating the recombinant peptide.

Alternatively, the polypeptide comprising, consisting or consisting essentially of an amino acid sequence corresponding to the sequence defined by SEQ ID NO: l or SEQ ID NO: 2 can be synthesised using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard in SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press at Oxford University, Oxford, England, 1989) or by Roberge et al. (1995 Science 269: 202). Syntheses may employ, for example, either t- butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) chemistries (see Chapter 9.1 of Coligan et al. supra; Stewart and Young, 1984, SOLID PHASE PEPTIDE SYNTHESIS, 2 ^nd ed. Pierce Chemical Co., Rockford, 111, 1994; and Atherton and Shephard, supra).

The AOS of the invention may be isolated from natural sources, or derived by chemical synthesis (for example, fmoc solid phase peptide synthesis as described in Fields G.B., Lauer- Fields J.L., Liu R.Q. and Barany G., (2002) Principles and Practice of Solid-Phase peptide Synthesis; Grant G., (2002) Evaluation of the Synthetic Product. Synthetic Peptides, A User's Guide, Grant G.A., Second Edition, 93-219; 220-291, Oxford University Press, New York) or genetic expression techniques. Standard recombinant DNA and molecular cloning techniques are described for example in Sambrook, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and Ausubel et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1987). The production of a protein or peptide for use in the invention by an appropriate transgenic animal, microbe, or plant is also contemplated.

The AOS may be connected to one or more additional compounds. For example, it may be connected to a compound that aids the function or activity of the AOS, protects the AOS from degradation, otherwise improves its half-life, aids in isolation and/or purification of the AOS during manufacture (for example ubiquitin, a His-tag, or biotin), or assists with cell membrane translocation or cell-specific targeting. These additional compounds may include, for example, peptides, nucleic acids, lipids and carbohydrates.

The additional compounds may be connected to the AOS, or synthesised as a part of a construct, using any appropriate means which allows the AOS to retain at least a level of its desired function. The term "connected" should be taken broadly to encompass any form of attachment, bonding, fusion or association between the AOS and the compound (for example, covalent bonding, ionic bonding, hydrogen bonding, aromatic stacking interactions, amide bonds, disulfide bonding, chelation) and should not be taken to imply a particular strength of connection. The AOS and the compound may be connected in an irreversible or a reversible manner, such that upon administration the AOS is released from the compound. EXAMPLES

EXAMPLE 1: ON-FARM TRIAL OVERVIEW

A large scale, statistically powered, trial was conducted on a commercial dairy farm to determine effects of Aloxsyn treated sperm/semen on the fertility of dairy cows following artificial insemination (AI).

The key scientific question to be addressed in the trial was - does treatment of bovine sperm with Aloxsyn during processing increase conception and pregnancy rates.

The overarching hypothesis is that Aloxsyn increases the resilience of bovine semen to processing procedures including dilution in extender, as well as freezing and thawing, thereby increasing its viability at AI and fertilisation of cycling dairy cows.

Aloxsyn is a variant of a plant enzyme produced by fermentation using a GMP applicable biotechnology manufacturing process. The enzyme is highly purified and formulated in a buffered solution.

Aloxsyn is unique in its ability to specifically target and neutralise lipid peroxides (LPOs). LPOs are toxic molecules formed when poly unsaturated fatty acids (PUFAs) are exposed to oxidative species. Bovine spermatozoa (as with oocytes) have a relatively high content of PUFAs in their cell membranes and are susceptible to oxidative damage owing to the oxidative environment they are exposed to during straw manufacture.

LPOs are particularly destructive to cells, acting to oxidise cytoplasmic proteins, cause mitochondrial dysfunction and damage DNA. Together these processes limit cell viability and initiate cell death processes.

Applicant's earlier work demonstrated that addition of Aloxsyn to fresh semen resulted in a 3.5-fold increase in progressively motile sperm 72h post collection compared to untreated semen (e.g. refer to WO 2015/183106). A second study demonstrated that addition of Aloxsyn to extended semen prior to chilling and freezing resulted in a 30% increase in the ratio of live progressively motile sperm to dead sperm following thawing. And finally, addition of Aloxsyn to semen straws after thawing resulted in a 50% increase in progressively motile sperm (e.g. refer to WO 2019/093909).

Together the above results evidence a beneficial effect of Aloxsyn on semen viability under conditions analogous to the process for semen straw manufacture and use. The Applicant hypothesised that Aloxsyn had the potential to either increase fertility when used at the standard semen dose per straw (15,000,000 sperm per straw) or to maintain fertility at a lower dose with either eventuality being of interest to the companies depending of the specifics of individual bull productivity.

The market for bovine semen is large with >50 million straws of bovine semen sold annually at prices of USD3 to USD20 per straw depending of the genetic merit of bulls for specific farm systems. The markets are largely concentrated in US, Europe, South America and Australasia and dominated by a few relatively large animal genetics interests including CRV (Netherlands), Genex (USA), Alta Genetics, Sexing Technologies, etc. Increasingly the market is also becoming differentiated by the use of sexed semen containing a much higher proportion of 'female' sperm. Importantly, the sexing process places additional oxidative stress on sperm and is also a potential target for Aloxsyn.

The intended primary outcome of the trial was to detect an increase in pregnancy rates with allene oxide synthase treatment as determined by pregnancy vet inspection at >80 days gestation. Effect of allene oxide synthase on calving rates was also monitored to determine effects on the quality of the pregnancy and calf health.

Allene oxide synthase was added to semen prior to freezing in the otherwise standard semen manufacturing process used by an animal genetics company partner. The allene oxide synthase treatment was present in the extended semen straws through a freeze/thaw process and at artificial insemination.

The following semen straws were used in the on-farm trial:

Table 1. Semen Straws for On-Farm Trial

Further, there were 5 bulls allocated across the treatments:

Table 2: Bulls used in On-Farm Trial

The commercial dairy farm operates using a continuous breeding, cow management system. The trial was based on a block design involving repeat breeding of cows on representation if they did not get pregnant. Individual animals received straws of each of the four treatment types from the same bull, in a pre-ascribed, random order, until they had been presented with all treatment alternatives or until they were pregnant. They were monitored for signs of pregnancy on an on-going basis. The on-farm information management system was used for both programming animal treatments and recording animal status. This information was downloaded at regular time intervals, compiled across time and rigorously validated before analysis. Data was analysed by an independent biostatistician using both descriptive statistics and generalised linear mixed models in Genstat.

Animals expected to come up for insemination during the trial had their 'Planned Sires' (PS; treatment and bull) block randomized and assigned in advance based on lactation and cycles to conception in the previous pregnancy. Each animal (CTL) was assigned to a specific bull and sequence (PS1, PS2, PS3, PS4) of treatments (see Table 3 below, as an example for 22 cows). This method was validated by an independent biostatistician.

Table 3: Example Animal/Treatment Randomisation

The trial was conducted in the cool winter (breeding) months of December to May thus avoiding the hot season associated with lower conception rates. Pregnancies were determined by ultrasound and palpation. These checks were divided into PI (38 days), Pl-B (60-days), P2 (90-120 days). Final trial numbers were determined by number of calves on the ground and their respective sires. EXAMPLE 2: ON-FARM PREGNANCY TRIAL RESULTS

Table 4 below summarises the breedings on trial.

Table 4:

Total breedings on trial were 8233. Of these 42 were removed because they were pregnant before trial start and 378 for various other reasons including assignment errors or because they related to dead or sold animals of unknown pregnancy status leaving 7791 breedings. The 7791 breedings were performed on 3579 trial cows. Thus, the trial cows received 4212 extra breedings. Of the 7791 breedings 425 were removed because they were to non-trial bulls leaving a total of 'on trial' breedings for further analysis of 7366. Short returns were omitted for the pregnancy analysis leaving 7366 - 833 = 6533 breedings for pregnancy analysis.

Preliminary analysis of the data was undertaken using descriptive statistics followed by more in-depth analysis using a fitted Generalised Linear Mixed Model (GLMM) to control for potential interactions between treatment and bull, cow age, cycle within lactation and breeding technician.

Generalised Linear Mixed Models in Genstat with binomial distribution and logit link were used to determine the proportion pregnant cows. The results are presented in Table 5 below, and should be read in conjunction with Figure 2. Treatment, Bull and their interaction, Cow age (3, 4, 5, 6, 7+) in years, Days from calving when bred ('48-75', '76-100', '101-150', '151+') were fitted as fixed effects.

Cow was specified as a random effect.

Back-transformed predicted means for Pregnant Rate on the percentage scale are presented. These are back transformed from predicted means on the logit scale.

Table 5: Confirmed Pregnancy Rates by Treatment Group

These data reveal that the two control groups achieved higher rates of pregnancy compared to the two AOS treatment groups. These results were unexpected. The standard error of the difference was 1.7%. A generalised linear mixed model analysis in Genstat was used to dissect interactions between treatments and other variables, including bull, cow age, days in milk (as an indicator of cycles required to achieve pregnancy), breeding technician and breeding type. Analysis of these factors could not explain the observed differences between treatment and control groups. Despite the between bull differences in fertility, no significant bull by treatment effect was observed (data not shown). This suggests that Aloxsyn was not preferentially affecting fertility of semen from some bulls compared to others. Slight increases in pregnancy rates were observed in 3 of 5 bulls when comparing the Optixcell + Aloxsyn treatment (Treatment Group 15) with its actual control (Optixcell Extender; Treatment Group 8) however these were never significant and not significant overall. Pregnancy rate showed the expected decrease with age, from 35.1% for 3-year olds to 26.5% for 7 years and older but this was not affected by treatment. Similarly, days in milk was found to have a significant effect on overall pregnancy rates being highest in animals in their second cycle (days 76 - 100) of the lactation compared to first cycle, third and fourth plus, but again, independently of treatment. While significant differences in breeding success of technicians was observed, the top 3, accounting for some 83% of breedings between them, had very similar performance (range 40 - 41.4%). Additionally, no relationship between treatment group pregnancy rates and technician was observed that might be considered to have impacted the results.

The final consideration was breeding type. Three breeding types were recorded in the data base (Standing, Timed AI, CN 4X Bred). By far the most common breeding type was 'Standing' where the animals had been through hormonal synchronisation programme and were adjudged to be on heat before breeding (5418 breedings). This group had a 43.5% pregnancy rate. The second group (termed 'Timed AT; 650 breedings) comprised animals that had been synched but were adjudged not to be on heat. These were frequently bred anyway for convenience but were found to have a significantly lower pregnancy rate (26%; P<0.001) compared to the Standing and CN 4X bred groups. The most likely explanation for this was that Timed AI animals were not actually 'on heat' at the time of breeding despite the synchronisation. The third group (CN 4X bred) is minor in number (198 breedings) and its unclear what part of the synchronisation cycle they were in but were adjudged to be on heat anyway at the time of breeding and so were bred. 41.5% of these breedings resulted in pregnancies. While marked differences in pregnancy rates were observed amongst breeding types, again there was no evidence of treatment related effects that may have affected the pregnancy results or their interpretation.

Collectively these data provided unequivocal evidence that AOS had no effect on increased pregnancy rate when used in an artificial insemination breeding program.

EXAMPLE 3: ON-FARM TRIAL CALVING RATES

In marked contrast to the pregnancy data, Aloxsyn was found to have a significant positive effect on the success of pregnancy assessed as 'live births'.

There were 2032 cows with known pregnancy outcomes and these were used for further analysis of successful calving. There were 2032 cows with known pregnancy outcomes: 1883 cows with singles and twins with at least one live calf and 149 cows with only dead calves or abortions. The percentage of known pregnancy outcomes with singles and twins with at Ieast one live calf was 95.0% for Treatment Group 15 (Optixcell + Aloxsyn) and 91.6% for Treatment Group 8 (Optixcell control); a 3.4% increase which was statistically significant (P Value = 0.040). The results are summarised in Table 6, below.

Table 6: Known pregnancy outcomes by treatment with P values for overall differences among various treatments

Moreover, the increased percentage of live births in the Aloxsyn treatment group was also trending to significance when compared to Treatment Group 3 (milk-based control). Note that pregnancy success is here calculated as percentage live births compared to confirmed pregnancy at P2. As P2 assessment occurs at 90 - 120d, Applicant's interpretation is that the positive Aloxsyn effect must be on established pregnancy which is surprising and yet potentially most beneficial.

Table 7: P Values for pairwise comparisons of Percentage Known Pregnancy outcomes with singles and twins with at least 1 Live calf by Treatment using Fisher's exact test

Approximately 5% of all calvings were recorded abortions and these did not differ significantly (P Value = 0.47) among treatments although it was lower in the Aloxsyn treatment group. This incidence is consistent with reported abortion rates. Similarly, about 84% of live births were normal size (consistent with expectations from the literature) and these did not differ significantly (P Value = 0.73) among treatments. Finally, 90% of live births had no problems with ease of birth and these did not differ significantly (P Value = 0.61) among treatments. The number of weak and deformed calves is very low across all four treatments.

Applicant concludes from these data that Aloxsyn had a significant positive effect on the overall success of pregnancy estimated as the percentage of viable live births. Moreover, taken in conjunction with the pregnancy data the successful calving picture provides compelling evidence that Aloxsyn is not only efficacious but safe to use in assisted reproductive applications as it had no measurable deleterious effect on the pregnancy.

In summary, 2032 cows with known pregnancy outcomes were used to determine treatment related effects on 'calves on the ground' and calf health. The results show that the AOS treatment group was successful in achieving a positive effect on calving. Conversely, this corresponded to a lower incidence of dead calves, abortions and other abnormalities with Aloxsyn treatment. The contention that Aloxsyn increases the success of pregnancy was further substantiated by analysis of the calf crop. Aloxsyn treatment was found to result in a substantial increase in calves categorised as 'large' (therefore more likely to thrive), but not 'very large' which is disadvantageous, compared to the other groups.

The intended purpose of this trial was to determine the effect of semen treatment with AOS on the eventual fertility of dairy cows at a scale and in a setting that was commercially meaningful to animal genetics company partners and dairy farmers. In perspective, fertility means different things to different people. To animal genetics companies it may mean increased pregnancies to semen, yet to the dairy farmer it may mean increased 'calves on the ground' because it is this that achieves herd replacement, genetic improvement and the next generation of milking animals. Increases in healthy 'calves on the ground' of 3.4% can be expected to translate into economic benefit for both the dairy farmer and animal genetics company. It is also likely that with more calvings the farmer is left with more cows in milk. With more calves and more milk per unit effort, farms will almost certainly become more profitable.

Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.