USE OF ALLENE OXIDE SYNTHASE FOR SEMEN PRESERVATION AND ASSISTED
REPRODUCTION
TECHNICAL FIELD
The invention relates to the use of the enzyme allene oxide synthase in the general
field of assisted reproductive technology. I n particular, the invention relates t o improving
the stability of semen (and sperm) for use in assisted fertilisation procedures including
artificial insemination, in vitro fertilisation, sperm sexing and in the In vitro manipulation of
oocytes in order to improve their viability and fertility. The invention relates to gametes
from humans, cattle, horses, pigs, poultry and other 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. These applications include the preservation
of plant seeds, the treatment of trauma patients with severe blood loss, the promotion of
apoptosis in the treatment of certain diseases, inducing resistance to herbicides in tobacco
plants, increasing the life span of sperm used for artificial insemination or in vitro
fertilisation, and for increasing the average and maximum life span of biological organisms.
However, these uses are entirely speculative having no experimental support in US
6,132,711.
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.
Additionally, antioxidants may be categorised as being of the 'suicide' variety
(meaning one molecule of antioxidant is consumed in neutralising one molecule of a
reactive oxidative species) or may be categorised as enzymic in which a single molecule of
enzyme may neutralise many oxidative molecules.
AOS has been shown to be of benefit in the treatment of severe ischemic injury and
Ischemia-reperfusion injury (US 7,157,082). Ischemic injury is caused by a traumatic
event, such as stroke or heart attack, which leads to a lack of blood supply causing a
shortage of oxygen to tissue. Ischemia-reperfusion refers to the tissue damage caused
when blood supply returns t o the tissue after a period of ischemia or lack of oxygen.
The stability of semen (as well as sperm and ova) 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
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 managed 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 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. These are often oxidative in nature
occurring as a result of thermal, pressure, light, chemical or biochemical insults to the cells
during processing.
The applicant has now found that AOS is an effective stability enhancer for both
fresh and frozen semen. Accordingly, it is an object of the invention t o provide a new way
t o extend the functional life of semen for use in assisted reproductive procedures for
humans and other animals, or at least to provide a useful alternative to existing methods.
SUMMARY OF THE INVENTION
I n one aspect the invention provides the use of allene oxide synthase as a
preservative for 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 preferably has 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 pHbuffered
aqueous medium. The allene oxide synthase may be present in the medium at any
suitable concentration, for example 1 to 10 g/m .
I n some embodiments of the invention, the semen is bovine semen. I n some other
embodiments of the invention, the semen is human semen. I n other embodiments, the
semen may be from a horse, sheep, pig or poultry.
I n a second aspect the invention provides a method of extending the life of semen by
contacting the semen with allene oxide synthase. Typically, the semen is stored in the
presence of allene oxide synthase.
I n certain embodiments of the invention, the allene oxide synthase is added to the
semen and the semen is frozen, stored for a period of time, and then thawed before use. I n
other embodiments, the semen is fresh semen.
Preferably the life of the semen is extended such that at least 50% of sperm in the
semen are viable 4 days from ejaculation.
I n a further aspect the invention provides semen containing allene oxide synthase.
The semen may be frozen or fresh.
I n another aspect the invention provides the use of the semen containing allene
oxide synthase In an artificial insemination process, an in vitro fertilisation process, or a
sperm sorting process. The artificial insemination process includes the step of artificially
inseminating a human female, a bovine cow, a horse, a pig, a sheep, or poultry.
I n yet another aspect the invention provides a composition for preserving semen
which comprises an effective amount of allene oxide synthase.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the mean relative survival of sperm in diluented bull semen stored
using standard 'fresh' methodologies for four days.
Figure 2 shows the effect of AOS on sperm motility of frozen bull semen.
Figure 3 shows the effect of AOS on sperm motility performance.
Figure 4 is the amino acid sequence SEQ ID No. 1.
DETAILED DESCRIPTION
The invention relates generally to the use of AOS as a preservative for semen. The
invention also relates to semen containing AOS as a preservative, to the use of the semen
containing AOS in an artificial insemination process, an in vitro fertilisation process, or a
sperm sorting process, and a composition for preserving semen which comprises AOS.
It will be appreciated that any reference in this specification to the preservation of
semen means extending its functional life beyond the functional life of untreated or
unprocessed semen, and includes the preservation of the sperm in that 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 embodiments 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 embodiments 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 al., 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 hydroxylor
sulphur-containing side chain, substitution of a n 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, 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 e t al. r Experiments with Gene Fusions, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1984); and Ausubel e 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.
Since AOS is an enzymatic antioxidant, it provides an additional benefit over
traditional chemical/non-enzymatic antioxidants, such as vitamin E, in that AOS is capable
of catalysing thousands of antioxidation reactions per AOS molecule. In contrast, most
chemical/non-enzymatic antioxidants only have the ability to take part in one reaction.
Consequently, AOS is effective at very low concentrations. Other antioxidants that must be
used at much higher concentrations (typically orders of magnitude higher) may have
toxicity problems.
Further, virtually all antioxidant enzymes, such as catalase and superoxide
dismutase, also generate a secondary pro-oxidant radical species that requires a second
enzyme t o remove it. AOS is believed to act against lipid hydroperoxides and is able to act
alone, i.e. in the absence of a second enzyme.
The applicant has found that AOS is an effective preservative of semen, both fresh
semen and frozen semen. AOS therefore has potential application in assisted reproduction
procedures including for humans and in animal breeding industries, particularly cattle and
horse breeding.
I n respect of the cattle breeding and dairy industries in New Zealand alone, it is
estimated that of 100 cows artificially inseminated 70 will not return and approximately 60
will be in calf. While various factors including infertility of the cows themselves may
account for the remaining 30 it is also widely recognised that semen quality is a key
consideration perhaps accounting for 10-15 of non-pregnant animals. In financial terms, a
1% gain in fertility is estimated to be worth $4M to the New Zealand dairy industry alone.
Progress with the infertility problem is therefore especially relevant to artificial insemination
and related semen processing industries such as those involved in X/Y sorting. I n addition
t o improving the pregnancy success rate for cows, improvements in semen quality can also
be expected t o result in cost benefits in other ways, such as a reduction in the number of
required dosages for fertilisation with concomitant increases in straws per ejaculate.
With respect to assisted reproductive technologies for humans, AOS may be useful
as an additive to ova culture media during in vitro fertilisation (IVF) procedures and for the
stabilisation of sperm prior to intracytoplasmic sperm injection (ICSI) extending to the
embryo culture, sorting, storage and transfer process.
While it is likely that there are several mechanisms of sperm ceil death in an animal
ejaculate, notably the two main ones, apoptosis and oxidative stress, each have lipid
peroxidation as a central step in the causal pathway. In these mechanisms oxidative
damage to lipid constituents of cell membranes including mitochondrial membranes results
in the loss of membrane integrity, cellular dysfunction and necrosis. Importantly, the
biochemistry of these processes involves oxidation of membrane lipids t o lipid peroxides
which then cascade on to cause oxidative damage of cellular proteins, DNA and other lipids.
This oxidative pathway t o cell death is also important in situations where cells are exposed
t o significant environmental challenges including heating/cooling, freezing/thawing, shear
forces and light exposure.
The role of oxidative damage has been recognised by industry for some time leading
to the development of antloxidative additives for semen and IVF diluents with some
success. One example Is Caprogen™ which is a protein extract of bovine liver containing a
catalase enzyme. The use of Caprogen™ is costly and, as a bovine offal extract, faces
restrictions in export markets.
The AOS will ordinarily be used in the form of an aqueous composition that includes
a buffer, and a semen extender. 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 1 to 10 g/ m , Alternative ranges include, but are not limited to, any range of
integers within 1 to 10 including any range having at it's lower limit 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 g/m and at it's upper limit 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g/ ml.
The applicant has determined that AOS was able to increase the survival of sperm in
fresh bull semen by ~50% after 4 days.
Example 1 shows that AOS is an effective preservative of fresh bull semen. At a
concentration of 3 Mg/ml, AOS increased the survival of sperm by approximately 50% after
4 days. An improvement of this order of magnitude is significant for several assisted
reproductive technology processes. It will be appreciated that the short 'shelf life' of fresh
untreated semen places significant constraints on its use especially if, for example, this first
requires transport before use, For this reason an Increase in sperm survival of 50% 4 days
after collection is economically important for cattle breeding because it markedly increases
the window of opportunity for use of the semen t o achieve its intended use of fertilising
cows, Similar improvements to the functional life of semen from other animals, including
humans, can be expected.
Example 2 confirms that AOS has a marked positive effect on maintaining sperm
motility post-thaw. The parameters examined include survival and motility post-thaw, and
enzyme dose/response effects. The applicant determined that the AOS significantly
increased post-thaw survival (P<0.05) and lifted motility by ~120% 24h post-thaw
(P<0.01) at an enzyme concentration of 2 g/m . Additionally, more sperm were found to
be membrane intact and available for fertilisation. Overall from the studies, it can be
concluded that AOS is effective at improving the tolerance of bovine sperm to dilution,
freezing and post-thaw processes. I n the case of cattle breeding, this means that many
more straws per ejaculate can be used thereby increasing the value of each ejaculate t o the
breeding industry.
Ovulation is a very discrete biological event in females, including dairy cows, and for
fertilisation t o occur the ova must encounter viable semen in the short period of its
functional life. I t will be appreciated that this is more likely to occur if the sperm retains its
motility for longer after insemination so as t o increase the probability that it is able t o
propel itself to the point of contact with the ova. Extended motility at 24 h is therefore a
strong indication that the overall viability (fertility) of AOS treated semen can be expected
to be greater. In cattle breeding, the number of animals that fail to become fertilised will
therefore be lower and this, in turn, can be expected to decrease costs and increase the
value of AOS treated semen,
Example 2 shows a decline in sperm motility (Figure 2) and in an index of sperm
motility performance (migratory efficiency, Figure 3) over time post-thaw. In particular, the
results for AOS at 2 g/ml show motility trending to significance across the time course. By
24 hours post-thaw, motility is more than double the control levels (+120%) and the
difference is highly significant (P<0.01), Additionally, the effect of AOS was shown to be
dose responsive and maximal at an enzyme concentration of 2 pg/ml. The positive effect of
AOS was even more pronounced in the Motility Index analyses (Figure 3), lifting this
measure of sperm performance by 2.9 fold after 24 hours.
AOS clearly has a significant positive effect on both sperm survival and measures of
sperm motility which are known key indicators of sperm performance and fertlity.
Additionally, more sperm were found to be membrane intact and available for fertilisation
following AOS treatment. At high dilutions (2 pg/ml), AOS was found to be effective at
improving the tolerance of bovine sperm to dilution, freezing and post-thaw processing in a
frozen semen process and the performance of semen in a fresh' process.
Example 3 shows that there was no effect on the total number of oocytes developing
to embryos, but there was a significant effect on the number of transferable quality
embryos produced from both the number of oocytes becoming mature and the number that
cleaved. The data indicate that AOS has no deleterious effects on oocyte developing to
embryos and increases the 'yield' of embryos that develop to maturity in IVF procedures.
As used in this specification, the words "comprises", "comprising", and similar words,
are not to be interpreted in an exclusive or exhaustive sense. In other words, they are
intended to mean "including, but not limited to".
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 - Fresh bull semen
This study was performed using ejaculates from 5 bulls using well established
procedures. Semen was collected using an artificial vagina at 42 °C and transferred to the
lab for initial quality assurance (volume, motility, sperm concentration). Samples were then
diluted to a standard sperm concentration of 10 X 10 spermatozoa per ml at 37 °C using
each of the experimental extenders. The experimental extenders comprised sodium citrate
1.856 g per 100ml, glucose 1 g per 100ml, egg yolk 20 ml per 100 ml containing antibiotic
preservatives, and were adjusted to pH 7.0. Experimental additives were then added t o this
extender base as shown in 1-5 below. Diluted samples (20 ml) were then cooled to 5 °C at
0.25 °C per minute and stored at 5 °C for 5 days, Samples were withdrawn at Day 1 and
Day 4 and percentage sperm survival and motility index determined using standard
procedures.
The results are shown in Figure 1, where:
1. Control - Diluent only
2. Catalase - Diluent plus 10 g/ ml catalase
3. Caprogen™ - Diluent plus Caprogen™ at standard concentration
4. AOS - Diluent plus 3 pg/ml AOS
5. AOS - Diluent plus 1 pg/ml AOS
Figure 1 shows the mean relative survival of sperm in diluented bull semen stored
using standard resh' methodologies for four days with and without test additives. Overall
control levels of survival at 4 days were ~55% of day 0 , The results are normalised against
the control. Thus, an AOS concentration of 3 pg/ml AOS results in a ~50% improvement in
survival compared t o the control. This difference is highly significant P<0.01. At an AOS
concentration of 1 pg/ml, the difference reached significance at the 0.05 level. The results
obtained with catalase and Caprogen™ were not significant.
Example 2 - Frozen bull semen
This study was conducted to determine whether AOS can improve the tolerance of
bovine sperm to the freeze-thaw procedure. Post-thaw motility was measured. Semen
samples from 5 bulls were diluted into media to standard concentration and each divided
into 2 aliquots. AOS was added to one aliquot to a final concentration of 2 pg/ml, with the
second aliquot from each bull acting as a control. The semen was the dispensed into straws
for freezing. The temperature of the straws was then reduced using standard procedures
and the straws stored in liquid nitrogen. After thawing at room temperature, the straws
were allowed to oxygenate using standard procedures. The straws were then stored at
room temperature for up t o 24 h prior to evaluation for survival, motility and motility index.
Sperm motility over time post-thaw is shown in Table 1 and Figure 2. % Motility
refers to the percentage of the total sperm showing movement. The control treatment is
the diluent and extender without AOS (Series 1). AOS treatment refers t o the diluent and
extender with 2 pg/ml AOS (Series 2). I n Figure 2, 1 is % motility pre-freezing, 2 is Ohr
post-thaw motility, 3 is 3hr post-thaw motility, 4 is 6hr post-thaw motility, and 5 is 24hr
post-thaw motility.
Table 1: Sperm motility
Motility index (relative proportion of motile sperm showing forward
propulsion) is shown In Table 2 and Figure 3. In Figure 3, 1 is Motility Index (MI) prefreezing,
2 is Ohr post-thaw MI, 3 is 3hr post-thaw MI, 4 is 6hr post-thaw M , and 5 is 24hr
post-thaw MI.
Table 2: Sperm motility index
Example 3 - I n vitro embryo culture
This experiment examined the effect of three concentrations (0.1 pg/ml, 1 pg/ml and
10 g/ m ) of AOS on embryo development and quality in an in vitro embryo production
system. Standard in vitro embryo production procedures were used for maturation,
fertilisation and culture of embryos. Small drops (20-50 L) of a medium comprising
embryo culture buffer or diluent in a petrie dish were overlaid with mineral oil. The initial
concentration of AOS in the medium was adjusted to give the correct final concentration
after the addition of oocytes, zygotes or embryos to the drops. Following culture, the
embryos were assessed morphologically at day 7, and the stage and grade of embryos were
recorded as per the IETS (International Embryo Transfer Society) manual for grading
embryos. The proportional data for development were analysed using Binomial Generalised
linear model. The embryo development data is summarised in Table 3 .
67
11
Table 3: Development of transferable grade VF embryos
1 = g AOS per ml medium
p <0.05
A significantly higher proportion of oocytes developed to transferable quality
embryos in the 1 g/ml AOS treatment group. The 0.1 pg/ml AOS had no effect on
development, while the higher level (10 pg/ml AOS) had a negative effect. The addition of
1 pg/ml of AOS to the culture medium had a beneficial effect on embryo quality, resulting in
a 7% increase in transferable embryos produced.
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.
CLAIMS
1. The use of allene oxide synthase as a preservative for semen.
2. The use as claimed in claim 1, wherein the allene oxide synthase is, or has
been cloned from, allene oxide synthase from Parthenium argentatum or is a functionally
equivalent variant thereof.
3. The use as claimed in claim 1 or claim 2, wherein the allene oxide synthase
has the amino acid sequence of SEQ ID No. 1 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 is in a pH-buffered aqueous medium.
5. The use as claimed in claim 4, wherein the allene oxide synthase is present in
the medium at a concentration in the range 1 to 10 g/ m .
6. The use as claimed in any one of claims 1 to 5, wherein the semen is bovine
semen.
7. The use as claimed in any one of claims 1 t o 5, wherein the semen is human
semen.
8 . A method of extending the life of semen comprising contacting the semen
with allene oxide synthase,
9 . A method as claimed in claim 8, wherein the semen is stored in the presence
of allene oxide synthase.
10. A method as claimed in claim 8 or claim 9, wherein allene oxide synthase is
added to the semen and the semen is then frozen.
11. A method as claimed in claim 10, wherein the frozen semen is thawed before
use.
12. A method as claimed in claim 9, wherein the semen is fresh semen.
13. A method as claimed in claim 12, wherein at least 50% of sperm in the
semen are viable 4 days from ejaculation.
14. Semen containing allene oxide synthase.
15. The semen as claimed in claim 14, which is frozen semen.
16. The semen as claimed in claim 14, which is fresh semen.
17. The use of the semen of any one of claims 14 to 16 in an artificial
insemination process, an in vitro fertilisation process, or a sperm sorting process.
18. The use as claimed in claim 17, where the artificial insemination process
includes the step of artificially inseminating a human female, a bovine cow, a horse, a pig, a
sheep, or poultry.
19. A composition for preserving semen which comprises allene oxide synthase.