WO2019093909A1

WO2019093909A1 - Allene oxide synthase for restoring or improving sperm viability

Info

Publication number: WO2019093909A1
Application number: PCT/NZ2018/050160
Authority: WO
Inventors: Mark Daniel BACKHAUS; Steven Charles Hodgkinson; Alexander Stanley HENNEBRY
Original assignee: Synthase Biotech Limited
Priority date: 2017-11-13
Filing date: 2018-11-13
Publication date: 2019-05-16

Abstract

The present invention relates to compositions, methods and uses involving an allene oxide synthase in the restoration or improvement of sperm viability, particularly for use in assisted reproductive procedures for humans and in the breeding of cattle, horses and other vertebrate animals. The allene oxide synthase may be used in an amount of 0.1 to 20 μg/mL, although according to certain examples described herein, the allene oxide synthase is used in an amount of 2-4 μg/mL. The allene oxide synthase may be obtained or derived from any species in which it is produced, including Parthenium argentatum, Parthenium argentatum, Solanum tuberosum, Solanum lycopersicum, Arabidopsis thaliana and Zea mays, and includes functional variants thereof produced by recombinant or synthetic means. In other examples according to the present invention the allene oxide synthase comprises and amino acid sequence defined by SEQ ID No: 1.

Description

ALLENE OXIDE SYNTHASE FOR RESTORING OR IMPROVING SPERM VIABILITY

TECHNICAL FIELD

The present invention relates to the use of the enzyme allene oxide synthase in restoring or improving sperm viability, for instance for use in assisted reproduction procedures including artificial insemination and in vitro fertilisation, especially in order to improve fertility. The present invention therefore provides compositions, methods and uses involving allene oxide synthase for restoring or improving the viability of sperm, and in particular sperm from humans, cattle, horses, pigs, poultry and all other vertebrate animals.

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. In the presence of AOS these compounds are rapidly converted to epoxides and the chain reaction is broken.

The ketol species produced by the action of AOS are relatively biologically inert compared to lipid peroxides and so US 6,132,711 speculates that the antioxidant effect of AOS may be useful in a variety of applications. Many agents, whether single compound or multiple ingredient substances, whether derived from natural biological sources or manufactured synthetically, are known to exhibit antioxidant behaviour. Some are broad spectrum antioxidants in that they exhibit an antioxidative effect in a range of biological systems, whereas others are selective and have an antioxidant effect in a specific set of biological parameters.

The stability of semen (as well as sperm) is widely recognised as a very significant constraint in the animal artificial insemination and assisted reproductive technologies sector. The instability of semen adds to cost, inefficiencies and logistical complexities. This is the case for both fresh semen and frozen semen, and across a range of species besides the bovine cattle breeding sector. Frozen sperm suffers from the apparent problem of loss of viability over the freezing and thawing steps, and fresh semen is severely constrained by a very limited shelf life (~3 days). Industry has attempted to manage the stability issue through a range of measures for improving efficiencies. For example, incremental improvements in diluent composition and processing are aimed at improving survival. Nonetheless, it is widely recognised that semen stability remains a major issue and there is an ongoing need to find ways to improve semen stability and thus performance. Additionally, the more advanced semen processing technologies, such as those involving sorting of sperm on the basis of whether they are X or Y (female or male), provide additional challenges to sperm viability as a result of the methodologies employed .

PCT international application published as WO2015/183016 describes the use of AOS as a preservative to be added to fresh semen, either prior to 'immediate' use (i.e. use within the limited shelf life, albeit extended by the addition of AOS) or prior to freezing of the semen for storage and use at a later time.

In work leading up to the present invention, the present inventors discovered that thawed straws of previously frozen bull semen contain a substantive population (up to 30%) of immotile sperm that are morphologically normal and were shown by the inventors to have all the characteristics of viable sperm, namely active mitochondria, intact membranes, and no evidence of DNA damage. The inventors then further discovered that these sperm can in fact be resuscitated upon the addition of AOS and thus deduced these sperm to be 'quiescent' rather than dead as previously believed . These initial experiments were performed on semen which had been frozen without the addition of AOS. The inventors then performed additional experiments to investigate whether this newly discovered post-thaw effect of adding AOS was also observed when AOS had already been added as a preservative agent prior to freezing of the semen. Interestingly, the inventors discovered that the viability of thawed sperm where AOS had been added prior to freezing was further improved upon addition of AOS post -thaw.

Accordingly, it is an object of the invention to provide a new approaches to restore or increase the viability of sperm, especially sperm of thawed semen, for use in assisted reproductive procedures for humans and other vertebrate animals, including to improve fertility, or at least to provide a useful alternative to existing methods.

SUMMARY OF THE INVENTION

The inventions described and claimed herein have many attributes and examples 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 examples identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.

In one aspect the present invention provides the use of allene oxide synthase in the restoration or improvement of sperm viability.

In another aspect the present invention provides a composition for restoring or improving the viability of sperm, the composition comprising sperm and an effective amount of allene oxide synthase. In another aspect the present invention provides a composition for restoring or improving the motility of sperm, the composition comprising sperm and an effective amount of allene oxide synthase.

In yet another aspect the present invention provides a composition for improving fertility in an assisted reproduction technique involving a human or non-human animal, the composition comprising sperm and an effective amount of allene oxide synthase.

In yet another aspect the present invention provides a method for restoring or improving the viability of sperm, the method comprising contacting sperm with allene oxide synthase in an amount sufficient to increase viability.

In yet another aspect the present invention provides a method for restoring or improving the motility of sperm, the method comprising contacting sperm with an allene oxide synthase in an amount sufficient to increase motility.

In yet a further aspect the present invention provides a method for improving fertility in an assisted reproduction technique involving a human or non-human animal, the method comprising contacting sperm with allene oxide synthase in an amount sufficient to increase fertility.

In some examples of these and other aspects of the present invention, the sperm comprises thawed semen.

In some examples of these and other aspects of the present invention, the sperm comprises human semen. In some other examples of these and other aspects of the present invention, the sperm comprises bovine semen. In some other examples of these and other aspects of the present invention, the sperm comprises equine semen. In other examples of these and other aspects of the present invention, the semen may be selected from the group consisting of ovine semen, caprine semen, porcine semen, and avian semen.

The allene oxide synthase may be, or may have been cloned from, allene oxide synthase from the guayule rubber plant Parthenium argentatum. The allene oxide synthase may have the amino acid sequence of SEQ ID No. 1 or is a functionally equivalent variant thereof.

The allene oxide synthase may be in any suitable form for use, for example in a pH- buffered aqueous medium. The allene oxide synthase may be present in the medium at any suitable concentration, for example 0.1 to 20 μg/mL.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the live/dead fluorescent staining and FACS separation results of Example 1, where Figure 1A shows the percentage (%) of live and dead cells in the absence (0) and in the presence ^g/ml) of AOS, where it can be seen that the percentage of live cells increased from 34.5 to 50.7% of total. Figure IB shows the live cells / dead cells with AOS treatment expressed relative to untreated, where it can be seen that when normalised against untreated, the ratio of live to dead cells was found to be increased by 47% following treatment with AOS. The error bars for both Figure 1A and Figure IB are SEM.

Figure 2 shows the sperm motility results by CASA analysis of Example 1 where Figure 2A shows the percentage (%) of total sperm demonstrating progressive motility in the absence (0) and in the presence ( lC^g/ml) of AOS, where it can be seen that the percentage of total sperm demonstrating motility was found to increase from 32.0 to 47.5% with AOS treatment. Figure 2B shows the relative progressive motility expressing treatment relative to untreated, where it can be seen that when normalised against untreated (D), the relative increase in motile sperm was found to be 48.4%. The error bars for both Figure 2A and Figure 2B are SEM.

Figure 3 shows the change in motility indices (ratios of different motility classes) between two different arms as described in Example 2, where the left bar shows the total number of progressive and slow progressive sperm (A+B) relative to the number of non- motile and immotile sperm (C+D) which can be expressed as ((A+B)/(C+D)). The right bar shows the total number of progressive and slow progressive and non-motile sperm (A+B+C) relative to the number of immotile sperm (D) which can be expressed as ((A+B+C)/D).

Figure 4 shows the amino acid sequences for various allene oxide enzymes derived from Parthenium argentatum (rubber plant; SEQ ID NO: 1); Solanum tuberosum (potato; SEQ ID NO: 2), Solanum lycopersicum (tomato; SEQ ID NO: 3), Arabidopsis thaliana (SEQ ID NO: 4) and Zea mays (corn; SEQ ID NO: 5).

Figure 5 shows the percentage of progressively motile sperm in treated and untreated semen samples post-thaw, at 0 and 8 hours, respectively.

DETAILED DESCRIPTION

In work leading up to the present invention, the present inventors discovered that thawed straws of previously frozen bull semen contain a substantive population (up to 30%) of immotile sperm that are morphologically normal and were shown by the inventors to have all the characteristics of viable sperm, namely active mitochondria, intact membranes, and no evidence of DNA damage. The inventors then further discovered that it was possible to rescue the viability of these sperm by contacting a semen sample with an allene oxide synthase, where it was observed that the amount of live sperm relative to dead sperm increased significantly (>30%) and where a significant increase in total sperm demonstrating progressive motility was also observed. To further illustrate this point, reference is made to Example 1 and Figures 1 and 2 which follow, where (i) an increase in the amount of live sperm relative to dead sperm and (ii) overall motility was observed for sperm treated with an allene oxide synthase. As such, the inventors concluded these sperm to be 'quiescent' rather than dead as previously believed. It was further discovered that addition of an allene oxide synthase also improved the viability of thawed sperm even where it had been previously exposed to an allene oxide synthase prior to freezing. Accordingly, the present invention relates generally to the use of allene oxide synthase (AOS) in the restoration or improvement of sperm viability.

It will be appreciated that any reference in this specification to the restoration or improvement of sperm viability may refer to an increase in the number or percentage of motile sperm. Sperm motility may be generally classified as follows: (A) progressive sperm; (B) slow progressive sperm; (C) non-motile sperm; and (D) immotile sperm. "Progressive sperm" may also be referred to as rapid progressive or fast progressive sperm, and may comprise sperm exhibiting a velocity of greater than 50 μη-ι/second. "Slow progressive sperm" may comprise sperm exhibiting a velocity of between about 10 to about 50 μη-ι/second. "Non-motile sperm" may comprise sperm exhibiting a velocity of between about 0 to about 10 μη-ι/second. "Immotile sperm" may comprise sperm exhibiting a velocity of between 0 μη-ι/second. Motile sperm as used herein may generally comprise progressive sperm and/or slow progressive sperm, and may or may not comprise non-motile sperm. Motile sperm may sometimes refer to all moving sperm. Reference to the restoration or improvement of sperm viability may also or alternatively refer to an increased motility index. The term "motility index" may refer to the number of progressive and/or slow progressive sperm relative to the number of non-motile and/or immotile sperm, or may refer to the number of progressive and/or slow progressive and/or non-motile sperm relative to the number of immotile sperm. For instance, a motility index may refer to (([A]+ [B])/([C]+ [D])), or to (([A]+ [B]+ [C])/[D]). Sperm motility may be measured by any method known in the art, including by way of Computer Assisted Sperm Analysis (CASA) systems.

Further, any reference in this specification to the restoration or improvement of sperm viability may also or alternatively refer to an increase in the number or proportion of live sperm cells relative to dead sperm cells. This may be determined using any method known in the art, including by way of flow cytometry (using staining and Fluorescent Activated Cell Sorting or FACS analysis).

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.

Accordingly, in one aspect the present invention provides a composition for restoring or improving the viability of sperm, the composition comprising sperm and an effective amount of allene oxide synthase.

In another aspect the present invention provides a composition for restoring or improving the motility of sperm, the composition comprising sperm and an effective amount of allene oxide synthase. It will be further appreciated that the restoration or improvement of sperm viability will lead to improved fertility. As used herein, the term "improving fertility" or "improvement in/of fertility" or similar refers to an improvement in the number, rate or percentage of conceptions, and/or an increase in the number, rate or percentage of pregnancies, and/or an increase in the number, rate or percentage of live births.

Accordingly, in another aspect the present invention provides a composition for improving fertility in an assisted reproduction technique involving a human or non-human animal, the composition comprising sperm and an effective amount of allene oxide synthase. In a related aspect, the present invention further provides a composition for improving fertility in a dairy cow, the composition comprising sperm and an effective amount of an allene oxide synthase.

In certain examples according to the compositions described herein, the allene oxide synthase is present in the composition at a concentration of between 0.1 and 20 μg/mL. The term "at a concentration of between 0.1 and 20 μg/mL" includes, without limitation, at a concentration of 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, 10.0, 10.2, 10.4, 10.6, 10.8, 11.0, 11.25, 11.5, 11.75, 12.0, 12.3, 12.6, 12.9, 13.0, 13.4, 13.8, 14.0, 14.5, 15.0, 15.33, 15.66, 16.0, 17.5, 18, 18.5, 19.0, 19.5 or 20 μg/mL allene oxide synthase, and includes any integer there between.

In other examples according to the compositions described herein, the allene oxide synthase is obtained or derived from, for example, Parthenium argentatum (rubber plant; SEQ ID NO: 1); Solanum tuberosum (potato; SEQ ID NO: 2), Solanum lycopersicum (tomato; SEQ ID NO: 3), Arabidopsis thaliana (SEQ ID NO: 4) and Zea mays (corn; SEQ ID NO: 5), and includes structural variant sequences which retain allene oxide synthase activity.

In certain other examples according to the compositions described herein, the allene oxide synthase comprises, or consists in, the amino acid sequence defined by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO : 5.

In some examples of the present invention, the sperm is compromised sperm or compromised semen. As used herein, the terms "compromised semen" and "compromised sperm" are intended to refer to semen or sperm which has undergone challenge or stress, and includes thawed semen or sperm. In some examples of the present invention, the sperm may comprise thawed sperm that has not previously been exposed to an allene oxide synthase. In some other examples of the present invention, the sperm may comprise thawed sperm that has been previously exposed to an allene oxide synthase. Thawed semen or thawed sperm refers to semen or sperm which was previously frozen, including by way of cryopreservation, and has been subsequently thawed, including for use in assisted reproductive technologies. Conventional wisdom is that cryopreservation of semen results in a high mortality rate of sperm but the use of thawed sperm has been essentially necessary due to the short shelf life of fresh semen. As such, semen has often been frozen in greater volumes and/or with higher concentrations of sperm to what is used in fresh semen to offset the apparent mortality levels of the sperm in the thawed semen. For instance, Vishwanath, R. Theriogenology, 59 (2003), 571-584, describes how cows are generally inseminated with about 10-20 x 10⁶ sperm when thawed sperm is used whereas equivalent fertilisation rates can be achieved with ~2 x 10⁶ of fresh sperm. The inventors have now shown that at least some of the sperm which was previously believed to be dead are in fact quiescent and can be remotilised by contact with AOS. As used herein, the term "quiescent sperm" refers to sperm which lacks motility but otherwise comprises the morphology of viable cells. Suitably the motility of quiescent sperm is able to be restored or improved upon the addition of AOS.

Accordingly, in another aspect the present invention provides a method for restoring or improving the viability of sperm, the method comprising the steps of:

a) providing a first composition comprising sperm; and

b) contacting the sperm with an allene oxide synthase;

to obtain a second composition comprising sperm having restored or improved viability compared to sperm not contacted with the allene oxide synthase.

Suitably, the first and/or the second composition comprises semen. Suitably, the first composition comprises thawed semen. In some examples of the present invention, the first composition comprises thawed semen that has not previously been exposed to an allene oxide synthase. In some other examples of the present invention, the first composition comprises thawed sperm that has previously been exposed to an allene oxide synthase.

Suitably, the first composition comprises quiescent sperm. Suitably, the first composition comprises at least 50% quiescent sperm, more suitably at least 60% quiescent sperm, even more suitably at least 70% quiescent sperm.

Suitably, the second composition comprises at least 40% viable sperm, more suitably at least 50% viable sperm, most suitably at least 60% viable sperm.

In some examples of the present invention, the second composition comprises at least 1% more viable sperm than the first composition. Suitably, the second composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% more viable sperm than the first composition.

In some examples, the second composition comprises a motility index that is at least 1% higher than the motility index of the first composition. Suitably, the motility index of the second composition is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50% higher than the motility index of the first composition.

In another aspect the present invention provides a method for restoring or improving the motility of sperm, the method comprising the steps of:

a) providing a first composition comprising sperm; and b) contacting the sperm with an allene oxide synthase;

to obtain a second composition comprising sperm having restored or improved motility compared to sperm not contacted with the allene oxide synthase.

Suitably, the first and/or the second composition comprises semen. Suitably, the first composition comprises thawed semen. In some examples, the first composition comprises thawed semen that has not previously been exposed to an allene oxide synthase. In some other examples of the present invention, the first composition comprises thawed sperm that has previously been exposed to an allene oxide synthase.

Suitably, the second composition comprises at least 40% motile sperm, more suitably at least 50% motile sperm, most suitably at least 60% motile sperm.

In some examples, the second composition comprises at least 1% more motile sperm than the first composition. Suitably, the second composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% more motile sperm than the first composition.

In yet another aspect the present invention provides a method for restoring or improving the viability of sperm, the method comprising the steps of:

a) providing a first composition comprising frozen semen comprising sperm;

b) thawing the first composition so as to obtain a second composition comprising thawed semen comprising sperm; and

c) contacting the second composition with an allene oxide synthase;

to obtain a third composition comprising sperm having restored or improved viability compared to sperm not contacted with the allene oxide synthase.

Suitably, the first and/or the second composition comprises semen. Suitably, the first composition comprises thawed semen. In some examples, the first composition comprises semen or sperm that has not previously been exposed to an allene oxide synthase. In some other examples, the first composition comprises semen or sperm that has previously been exposed to an allene oxide synthase.

Suitably, the second composition comprises quiescent sperm. Suitably, the second composition comprises at least 50% quiescent sperm, more suitably at least 60% quiescent sperm, even more suitably at least 70% quiescent sperm.

Suitably, the third composition comprises at least 40% viable sperm, more suitably at least 50% viable sperm, most suitably at least 60% viable sperm. In some examples, the third composition comprises at least 1% more viable sperm than the second composition. Suitably, the third composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% more viable sperm than the second composition.

In some examples, the third composition comprises a motility index that is at least 1% higher than the motility index of the second composition. Suitably, the motility index of the third composition is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50% higher than the motility index of the second composition.

In yet another aspect the present invention provides a method for restoring or improving the motility of sperm, the method comprising the steps of:

a) providing a first composition comprising frozen semen comprising sperm;

c) contacting the second composition with an allene oxide synthase;

to obtain a third composition comprising sperm having restored or improved motility compared to sperm not contacted with the allene oxide synthase.

Suitably, the third composition comprises at least 40% motile sperm, more suitably at least 50% motile sperm, most suitably at least 60% motile sperm.

In some examples, the third composition comprises at least 1% more motile sperm than the second composition. Suitably, the third composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% more motile sperm than the second composition.

The term "compromised semen" or "compromised sperm" may also refer to semen or sperm which has undergone challenge or stress due to other processing techniques. For instance, semen processing technologies, such as those involving in the sorting of sperm on the basis of whether they are X or Y (female or male) to as to produce sexed semen appear to provide additional challenges to sperm viability as a result of the methodologies employed. Accordingly, compromised semen or sperm may also include sexed semen or sperm. The term "compromised semen" or "compromised sperm" and similar as used herein may also refer to semen exposed to oxidative stress, hydrodynamic shear forces, changes in osmolality, and/or changes in pH.

In some examples of the present invention, the sperm comprises human semen. In some other examples of the present invention, the sperm comprises bovine semen. In some other examples of the present invention, the sperm comprises equine semen. In other examples of the present invention, the semen may be selected from the group consisting of ovine semen, caprine semen, porcine semen, and avian semen .

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 the allene oxide synthase isolated from the rubber plant Parthenium argentatum. The term also includes 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 "functionally equivalent variant" as used in this specification includes those peptides or proteins having one or more (for example 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5) deletions, additions and/or substitutions while substantially retaining the desired function of the AOS or to variants that are derivatised by chemical modification of selected amino acids or the overall amino acid structure. Amino acid substitutions will typically be conservative amino acid substitutions. It should be appreciated that a functionally equivalent variant may have a level of activity higher or lower than the protein of which it is a variant. In various examples of the invention, a functionally equivalent variant has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the level of activity of the protein of which it is a variant. Functionally equivalent variants will have antioxidant activity. For example, they may have the ability to convert lipid peroxides/hydroperoxides to lipid epoxides at the lipid/cell membrane interface.

In some examples of the invention, the AOS is the AOS described in Genbank CAA55025.2 or a functionally equivalent variant of this protein.

Those skilled in the field will readily be able to assess the function and determine the level of activity of a protein based on the information in this specification and using known techniques. By way of example, antioxidant activity can be determined using the methods described in Pinchuk et a/., Chemistry and Physics of Lipids, 164 (2001), 42-48, or using a commercially available assay kit available thought Sigma-Aldrich.

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 ami no 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, theronine, 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 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 a/., 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 particula r 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.

The AOS may be in any suitable form for use, including in the form of a pH-buffered aqueous medium. The extender typically consists of a diluent buffer, protein and lipid components. The AOS may be present at any suitable concentration, for example in the range 0.1 to 20 μg/mL. Alternative ranges include, but are not limited to, any range of integers within 0.1 to 20 including any range having at its lower limit 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg/mL and at its upper limit 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg/mL. Suitably, the AOS is present at a concentration of 2 μg/mL. More suitably, the AOS is present at a concentration of 4 μg/mL.

The inventors have found that AOS can be used to remotilise quiescent sperm, which can result from processing semen during assisted reproduction procedures. AOS therefore has potential application in assisted reproduction procedures including for humans and in animal breeding industries, particularly bovine (including but not limited to cattle and water buffalo), equine, ovine, caprine, porcine and avian (including but not limited to poultry such as turkey or chicken) breeding.

Accordingly, in another aspect the invention provides the use of an allene oxide synthase in the restoration or improvement of sperm viability for use in an artificial insemination process or an in vitro fertilisation process. The artificial insemination process includes the step of artificially inseminating a female human, a cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken) with the sperm. The in vitro fertilisation process includes the step of mixing the sperm with the egg.

In yet another aspect the invention provides a method for restoring or improving sperm viability for use in an artificial insemination process or an in vitro fertilisation process, the method comprising contacting the sperm with allene oxide synthase. The artificial insemination process includes the step of artificially inseminating a female human, a cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken) with the sperm. The in vitro fertilisation process includes the step of adding the sperm to the egg .

Accordingly, in another aspect the present invention provides a method for artificial insemination of a female animal, the method comprising the steps of:

a) providing sperm;

b) contacting the sperm with an allene oxide synthase; and

c) artificially inseminating the female animal with the contacted sperm from b). Suitably, the sperm provided in step a) comprises thawed semen.

Suitably, the female animal comprises a human female, a bovine cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken) .

In yet another aspect the present invention provides a method for artificial insemination of a female animal, the method comprising the steps of: a) providing frozen semen comprising sperm;

b) thawing the frozen semen so as to obtain thawed semen comprising sperm;

c) contacting the sperm with allene oxide synthase; and

d) artificially inseminating the female animal with the contacted sperm from c). Suitably, the female animal comprises a human female, a bovine cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken) .

In the assisted reproduction technologies for both humans and commercial animals, it is widely recognised that sperm viability is a key consideration perhaps accounting for low or dropping fertility rates.

Accordingly, in another aspect the present invention provides a method for improving fertility of a female animal, the method comprising the steps of:

a) providing sperm;

b) contacting the sperm with allene oxide synthase; and

Suitably, the female animal comprises a human female, a bovine cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken).

In yet another aspect the present invention provides a method for improving fertility of a female animal, the method comprising the steps of:

a) providing frozen semen comprising sperm;

b) thawing the frozen semen so as to obtain thawed semen comprising sperm;

c) contacting the sperm with allene oxide synthase; and

d) artificially inseminating the female animal with the contacted sperm from c). Suitably, the female animal comprises a human female, a bovine cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird (e.g. a turkey or a chicken).

The Applicants recognise that the compositions and methods described herein are particularly useful in improving the fertility of dairy cows in a herd. In Example 4, which follows, the results of an on-farm artificial insemination trial involving a dairy herd is reported. These data show encouraging rates of fertility in dairy cows adopting the compositions and methods described herein.

Accordingly, in another aspect the present invention provides a method for improving fertility of a dairy cow, the method comprising the steps of:

a) providing sperm;

b) contacting the sperm with allene oxide synthase; and

c) artificially inseminating the dairy cow with the contacted sperm from b).

Suitably, the sperm provided in step a) comprises thawed semen.

In yet another aspect the present invention provides a method for improving fertility of a dairy cow, the method comprising the steps of:

a) providing frozen semen comprising sperm; b) thawing the frozen semen so as to obtain thawed semen comprising sperm;

c) contacting the sperm with allene oxide synthase; and

d) artificially inseminating the female animal with the contacted sperm from c).

In some examples according to the methods described herein, the sperm is contacted with an allene oxide synthase at a concentration of between 0.1 and 20 μg/mL. The term "at a concentration of between 0.1 and 20 μg/mL" includes, without limitation, at a concentration of 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, 10.0, 10.2, 10.4, 10.6, 10.8, 11.0, 11.25, 11.5, 11.75, 12.0, 12.3, 12.6, 12.9, 13.0, 13.4, 13.8, 14.0, 14.5, 15.0, 15.33, 15.66, 16.0, 17.5, 18, 18.5, 19.0, 19.5 or 20 μg/mL allene oxide synthase, and includes any integer there between.

In other examples according to the methods described herein, the allene oxide synthase is obtained or derived from, for example, Parthenium argentatum (rubber plant; SEQ ID NO: 1); Solarium tuberosum (potato; SEQ ID NO: 2), Solarium lycopersicum (tomato; SEQ ID NO: 3), Arabidopsis thaliana (SEQ ID NO: 4) and Zea mays (corn; SEQ ID NO: 5), and includes structural variant sequences which retain allene oxide synthase activity.

In certain other examples according to the methods described herein, the allene oxide synthase comprises, or consists in, the amino acid sequence defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

In addition to improving fertilisation success rates, improvements in sperm viability can also be expected to result in benefits in other ways. For instance, in commercial animals, the improved numbers of viable sperm may permit a reduction in the volume of semen required for each artificial insemination. This can be especially desirable for rare or valuable semen, for example from high breeding worth animals. Furthermore, in some instances semen is concentrated prior to freezing in order to reduce its volume (and increase the concentration of sperm in the semen). The concentration process may stress the sperm and the improvement in sperm viability provided by the present invention may mitigate the need to concentrate the semen prior to freezing thus also reducing the stress on the sperm.

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 (for example, in protein chemistry and biochemistry).

It is intended that reference to a range of numbers disclosed herein (e.g. 1 to 10) also incorporates reference to all related numbers within that range (e.g. 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.

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.

The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field.

EXAMPLES

Example 1 - Post-thaw addition of AOS to semen Live/dead staining

To determine the effect of the post-thaw addition of AOS on the proportion of live versus dead bull semen, 2 straws containing frozen bull semen from a single bull were thawed by immersing in a 37 °C water bath for 30 seconds, after which AOS (3 μg/mL) was added to half of the volume (125 μΙ_) of each of the semen straws. The semen samples in the absence and presence of AOS were then stained using the ThermoFisher Scientific LIVE/DEAD Sperm Viability Kit (L7011) as per the manufacturer's instructions. Following staining, Fluorescent Activated Cell Sorting (FACS) analysis was performed using a BD FACS Verse™ flow cytometer. The relative proportion of live cells was then determined based on the levels of green (live cell) and red (dead cell) fluorescence of 10,000 sperm cells.

The results are shown in Figure 1, where Figure 1A shows the percentage (%) of live and dead cells as determined by Invitrogen PI Cybrl4 fluorescent staining and FACS separation in the absence (0) and in the presence (3 μg/mL) of AOS, where it can be seen that the percentage of live cells increased from 34.5 to 50.7% of total. Figure IB shows the live cells / dead cells with AOS treatment expressed relative to untreated as determined by the staining for Figure 1A, where it can be seen that when normalised against untreated, the ratio of live to dead cells was found to be increased by 47% following treatment with AOS. The error bars for both Figure 1A and Figure IB are SEM. Motility

To determine the effect of the post-thaw addition of AOS on the motility of bull semen, AOS (10 μg/mL) was added to half of the volume of a semen straw immediately post-thaw (37 °C water bath for 30 seconds) with the other half of the straw being used as the untreated control. Samples were mixed well and a 10 μΙ_ aliquot was used to assess semen motility in the absence and presence of AOS using a Biophos Qualisperm Computer Assisted Sperm Analysis (CASA) system and Leija slides.

The results are shown in Figure 2, where Figure 2A shows the percentage (%) of total sperm demonstrating progressive motility as determined by CASA in the absence (0) and in the presence (10 μg/mL) of AOS, where it can be seen that the percentage of total sperm demonstrating motility was found to increase from 32.0 to 47.5% with AOS treatment. Figure 2B shows the relative progressive motility as determined by CASA and expressing treatment relative to untreated, where it can be seen that when normalised against untreated (D), the relative increase in motile sperm was found to be 48.4%. The error bars for both Figure 2A and Figure 2B are SEM.

Example 1 thus demonstrated that addition of AOS improved the percentage of live cells and the percentage of motile sperm within semen.

Example 2 - Addition of AOS to semen pre-freeze and post-thaw

In this example, semen from four bulls was used where for each bull, three straws for two separate collects (ejaculates) were used, thus resulting in 24 tests for each arm.

2 μg/mL of AOS was added to each straw prior to freezing. The straws were then thawed and had either 0 μg/mL (first arm) or 2 μg/mL of AOS (second arm) added, so that the arms received the following treatments:

(1) First arm : 2 μg pre-freeze + 0 μg post-freeze; and

(2) Second arm: 2 μg pre-freeze + 2 μg post-freeze.

The motility of the sperm within each arm was then measured by CASA.

The results are shown in Figure 3, where Figure 3 shows the change in motility indices between the two different arms. The motility indices used in Figure 3 illustrate differences between combined motility classes where the classes are referred to using the following letters:

A refers to progressive sperm;

B refers to slow progressive sperm;

C refers to non-motile sperm; and

D refers to immotile sperm.

In Figure 3, the left bar shows the total number of progressive and slow progressive sperm (A+B) relative to the number of non-motile and immotile sperm (C+D) which can be expressed as ((A+B)/(C+D)). The right bar shows the total number of progressive and slow progressive and non-motile sperm (A+B+C) relative to the number of immotile sperm (D) which can be expressed as ((A+B+C)/D).

An increase in the motility index illustrates a decrease in the non-motile combined with immotile populations ((A+B)/(C+D)) or the immotile population alone ((A+B+C)/D). The increase observed in Figure 3 is an increase in the indices of the second arm relative to the indices for the first arm and indicates further movement of the semen from immotile to motile classes.

Example 3: Additional Post-Thaw Data

Semen straws: 15 million sperm per 0.25 mL straw, extended in OPTIXcell and stored frozen in liquid nitrogen before use. The OPTIXcell was obtained from IMV Technologies Ltd and prepared for use according to the manufacturer's instructions. Details of the semen cooling and freezing process are provided below.

Allene oxide synthase (SEQ ID NO: 1) was manufactured according to standardised procedures by Synthase Biotech Ltd and was of >95% purity. The material was stored at - 20 °C before use and thawed at room temperature for approximately 30 minutes with occasional gentle mixing before addition to samples.

Semen straws (3 bulls, 7 straws) were thawed in a ~ 30 °C water bath and remained at ~30-35 °C during sample preparation and analysis.

To conduct experiments aliquots of IVF media, either containing allene oxide synthase

(5 ug/mL final; treatment) or no allene oxide synthase (vehicle [buffer only]; control) were added to semen samples and incubated for the indicated time periods. The semen samples and media were mixed in equal proportions.

An aliquot of each sample was then subjected to CASA analysis at (0 h) and following 8 h incubation at 30-35 °C.

The results are presented in Figure 5 and show the percentage of progressively motile sperm in samples. Progressive motility is presented because it is a widely accepted proxy for the viability/fertility of semen. Immediately post-thaw the control sample showed ~44% progressive motility falling to ~15% and ~25% for the control and allene oxide synthase treated samples respectively following 8 h incubation at 30-35 °C. The difference between the 8 h control and 8 h allene oxide synthase treated sample is highly significant (p<0.02).

The data evidences protective and/or restorative effects of allene oxide synthase on the physiological challenge of semen freeze / thawing and post thaw motility. For example, the 8h allene oxide synthase treated sample retained >55% of Oh progressive motility. Resilience to 'stress testing' of semen by extended incubation at 30-35 °C has been used by industry as a proxy for fertility. The test would normally use 3 h incubation because this is often considered the cut off for viability in in vitro testing. For a sample to retain >50% of 0 h motility following 8h incubation is a surprising finding therefore and illustrates the efficacy of allene oxide synthase at extending viability of sperm post thaw. Example 4: On-Farm Artificial Insemination Trial

Semen and Activator Straw Manufacture & Use

Semen straw: 15 million sperm per 0.25 mL straw extended in OPTIXcell and stored frozen in liquid nitrogen (LN2) before use. The semen used in this study was from 5 production bulls and collected using standard procedures for a bovine animal genetics company.

Activator: OPTIXcell extender containing allene oxide synthase at 4ug/ml_ in a 0.25 mL straw. Also stored frozen in liquid nitrogen. Note, the Activator straw does not contain any sperm cells.

Allene oxide synthase (SEQ ID NO: 1) was manufactured according to standardised procedures by Synthase Biotech Ltd and was of >95% purity. The material was stored at - 20 °C until the day of use and thawed at room temperature for approximately 30 minutes with occasional gentle mixing before addition to OPTIXcell.

OPTIXcell was obtained from IMV Technologies Ltd and prepared for use according to manufacturer's instructions. It was treated with CSS GTLS antibiotic cocktail and warmed to 35 °C before allene oxide synthase addition to a concentration of 4 ug/mL.

For Activators, extender was dispensed directly into straws. For semen straws the extender was diluted with bull ejaculate, 1 : 1 and incubated for 10 min at 35 °C. Straws were then chilled in a water jacket to 4 °C prior to being frozen in an IMV Digitcool freezer following the IMV freeze curve programme down to -140 °C.

Straws were then stored in LN2 tanks prior to shipping to the dairy farm for the trial. Care was taken to ensure that straws remained frozen at LN2 temperature until the time of use. Straws selected for an AI were run through a 45 second warm water bath procedure and held in a 'gun' warmer (pistolette) until insemination. To avoid variability in sperm viability, a 15-minute window was adhered to between straw thawing and insemination.

On-Farm Trial

An on-farm dairy cow fertility trial was carried out to determine the effects of allene oxide synthase on the conception rate of dairy cows undergoing artificial insemination (AI) with frozen semen. The study herd was a selection of multiparous lactating cows, a subset of which were inseminated using the allene oxide synthase treatment.

The cows entered a reproduction oestrus synchronisation programme 50 days after their last calving. The sync program consisted of administering reproductive hormone treatment at prescribed intervals until the animal entered heat (underwent ovulation) where upon it was subjected to AI. Consistent with normal farm practice, this programme of synching and AI was continued until animals fell pregnant.

Allene oxide synthase treatment inseminations consist of a variation of the standard AI practice in which cows were administered firstly, a straw containing allene oxide synthase (the Activator straw) and subsequently a semen containing straw. The administrations were performed by trained AI technicians using techniques well established in the dairy industry and consisted of administering the contents of the straws into the neck of the cervix using a pistolette device. In practice two pistolettes were used, one for the Activator straw and subsequently, the semen containing straw.

Pregnancies were assessed by palpation and ultrasound scanning at 38 days, 60 days, and 90-120 days post AI. The 90-day scan was performed by a registered veterinarian.

Results of the trial to date are shown in Table I as follows.

Table I: On- Farm Trial Data

*last pregnant cow due to give birth 27/2/ 19

The data shows that 1643 animals received the allene oxide synthase treatment resulting in 575 confirmed pregnancies. As at October 2018 (~a quarter of the way through calving), the pregnancies had resulted in 114 live births with the health of calves at birth being consistent with the norm for the farm.

Taken together, the data demonstrates the principle that it is possible to treat pre- frozen semen with allene oxide synthase at the time of AI and that this can beneficially be achieved via a second straw. Animals treated this way become pregnant and the pregnancies result in live births.

The reason the current example is relevant and important is that animal genetics companies frequently hold stocks of frozen semen from animals of high genetic merit and / or low fertility which are of special value. Treatment with allene oxide synthase after thawing provides a mechanism for imparting the benefit of the enzyme to semen that was not manufactured containing it. For semen that was not manufactured contain ing allene oxide synthase, post thaw addition therefore represents an approach to increase semen motility and dairy cow conception.

Post thaw addition of allene oxide synthase is a novel and non-obvious approach to improving semen viability and the fertility of dairy cows. To the inventors' knowledge, no other examples of post thaw treatments being administered to AI semen with the objective of improving semen performance have been reported to date. Consequently, the approach used here, and findings are most surprising.

_*** All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications and variations are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as described herein, and as defined by the appended claims.

Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification. The specific compositions and methods described herein are representative of preferred examples and are exemplary and not intended as limitations on the scope of the invention. Other aspects and examples will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed as essential. Thus, for example, in each instance described or used herein, in examples or examples of the present invention, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms in the specification. Also, the terms "comprising", "including", containing", etc. are to be read expansively and without limitation, meaning that these terms 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.

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. Further, as used or described herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or examples or methods specifically disclosed herein.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other examples are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. The use of an allene oxide synthase in the improvement or restoration of sperm viability.

2. The use as claimed in claim 1, wherein the allene oxide synthase comprises, or has been cloned from, allene oxide synthase from Parthenium argentatum, Parthenium argentatum, Solanum tuberosum, Solanum lycopersicum, Arabidopsis thaliana and Zea mays or comprises a functionally equivalent variant thereof.

3. The use as claimed in claim 1 or claim 2, wherein the allene oxide synthase comprises the amino acid sequence of any one of SEQ ID Nos. 1-5 or is a functionally equivalent variant thereof.

4. The use as claimed in any one of claims 1 to 3, wherein the allene oxide synthase comprises the amino acid sequence of SEQ ID No: 1.

5. The use as claimed in any one of claims 1 to 4, wherein the allene oxide synthase is in a pH-buffered aqueous medium.

6. The use as claimed in claim 5, wherein the allene oxide synthase is present in the medium at a concentration in the range 0.1 to 20 μς/ιηΙ.

7. The use as claimed in any one of claims 1 to 6, wherein the sperm comprises thawed semen.

8. The use as claimed in any one of claims 1 to 7, wherein the semen comprises human semen, bovine semen, or equine semen.

9. A composition for restoring or improving sperm viability which comprises sperm and an effective amount of allene oxide synthase.

10. The composition as claimed in claim 9, wherein the semen is thawed semen.

11. A method for restoring or improving sperm viability, the method comprising contacting the sperm with allene oxide synthase.

12. The method of claim 11, wherein the semen comprises thawed semen.

13. A method for artificial insemination of a female animal, the method comprising the steps of: a) providing sperm;

b) contacting the sperm with an allene oxide synthase;

c) artificially inseminating the female animal with the contacted sperm from b).

14. The method as claimed in claim 13, wherein the semen comprises thawed semen.

15. The method as claimed in claim 13 or claim 14, wherein the female animal comprises a human female, a bovine cow, a water buffalo, a horse, a sheep, a goat, a pig, or a bird.