EP4680334A1

EP4680334A1 - An in vitro method for enhancing the effective functionality of reproductive cells

Info

Publication number: EP4680334A1
Application number: EP23703705.6A
Authority: EP
Inventors: Desmond Patrick LEADON
Original assignee: Desmond P Leadon Ltd
Current assignee: Desmond P Leadon Ltd
Priority date: 2023-01-31
Filing date: 2023-01-31
Publication date: 2026-01-21
Also published as: WO2024160352A1

Abstract

An in vitro method for enhancing the effective functionality of a reproductive cell, comprises supplementing the endogenous ubiquinol of the cell by contacting a medium containing said reproductive cell with an aqueous solution in which ubiquinol is uniformly dispersed. The method has particular application in the assisted reproduction of human and non-human animals including enhancing the fertility of animals used for food production for humans and other animals, by increasing litter size and/or conceptions/ejaculate and decreasing the period between parturition and the next/subsequent conception, and, thereby, overall livestock production rates stemming from artificial or assisted reproductive technologies.

Description

An in vitro method for enhancing the effective functionality of reproductive cells

Field of the Invention

This invention relates to an in vitro method for enhancing the effective functionality of reproductive cells and, in particular, to an in vitro method for enhancing the effective functionality of reproductive cells of both human and non-human animals.

There are many reasons why there is a need for enhancing the effective functionality of reproductive cells in assisted reproduction techniques.

Background of the Invention

In the case of humans, 1 in 6 couples nowadays are sub-fertile, i.e., 100 million couples worldwide according to statistics provided by the World Health Organisation (WHO). Thus, many couples are experiencing infertility and reproductive dysfunction for a variety of reasons, many are linked to oxidative stress, egg health and sperm health and motility. As a result, the subjects require professional assistance with reproduction.

Assisted reproduction includes laboratory techniques in which extended, preserved or enhanced semen is made available for insertion into a female or a female’s ovum or ova. In the case of humans, one of several different methods of sperm preparation are utilised to enhance compromised semen for insertion into a woman.

In the case of non-human animals, semen extenders are used to cool or freeze animal semen for transport and insemination of multiple females, as required. For example, a freezing medium for bull semen is marketed under the Trade Mark OptiXcell by the company IMV Technologies.

In many cases the age of one or both partners of a human couple is a factor. However, failure to conceive is frequently due to subfertility in the male partner; 40% of such cases are due to male factors.

Formerly, 35% sperm motility was considered as the threshold for substandard or unacceptable motility in men. That figure has now been reduced to 25%.

In the case of non-human animals, other reasons for enhancing fertility include, for example, enhancing the fertility of animals used for food production for humans and other animals, such that increasing litter size and, thereby, livestock production rates, would be advantageous.

In breeding programs involving artificial insemination (Al), sperm from elite males is used, so there is a need for the potency of sperm to be maximised for such programs.

Any laboratory interference/processing of sub-fertile human sperm cells challenges their already compromised viability. Laboratory interference with normal animal sperm cells, for example separating them from their own seminal fluids and substituting these fluids with semen extenders and then cooling them and/or freezing them for later use in the female, also compromises their viability.

All sperm cells, whether human or non-human animal cells, require considerable energy in their highly competitive journey from release to the achievement of conception. The related energy production results in the release of reactive oxygen species (ROS) and excess ROS results in oxidative stress.

Thus, the enhancement of sperm cell motility in assisted reproduction techniques is often a crucial factor in achieving a successful outcome.

ROS are counteracted by antioxidants of which there are many.

In the case of sperm, an increase in ROS results in oxidative stress and a decrease in sperm motility due to damage to the cell membrane, cells and damage to the DNA in the sperm nucleus.

Antioxidants are currently used, for example, in the manufacture of human sperm preparations by a number of companies, for example the company Vitrolife markets a fertilisation medium which has triple antioxidant protection in the form of a combination of acetyl-L-camitine, a-lipoic acid and N-acetyl-L-cysteine.

Coenzyme Q10 (CoQlO) is a very powerful, naturally occurring antioxidant which exists in two forms, namely ubiquinone and ubiquinol, and every living cell relies on it for energy production and protection from oxidative stress. The most biologically active form of CoQlO is the reduced form ubiquinol, which is eight times more potent than ubiquinone. Ubiquinone is converted intracellularly to ubiquinol, for example in the process of the aerobic production of the energy needed for sperm motility.

Although about 95% of circulating CoQlO is in the form of ubiquinol, both forms of CoQlO are present in the mitochondrial membrane of every cell in the body. Ubiquinone is required for the mitochondrial ATP synthesis which is the source of the cellular energy that allows the sperm to move. In the course of catalysing this reaction, ubiquinone is converted to the reduced form of CoQlO, ubiquinol. The anti-oxidant effect of ubiquinol is essential to the effective movement of sperm because, even if sufficient ATP exists, oxidative stress can damage the integrity of the cell membrane of the sperm thereby interfering with its ability to move effectively or progressively. It is for this reason that emphasis is placed on progressive motility when assessing semen quality as aberrant sperm motion is of no consequence to potential fertilisation, even if they are motile.

CoQlO, including the reduced form ubiquinol, when administered orally as a dietary supplement to humans has been shown to improve both egg and sperm health and, in turn, embryo quality. However, very large oral doses are required over a prolonged time to achieve minimal increases in CoQlO in sperm cells.

CoQlO is also used as an adjunct to statins in the treatment of cardiac disease. Direct administration of CoQlO (rather than oral administration) to sperm cell fluids and egg /follicular fluids has also been tried to equivocal effect.

Furthermore, direct administration of CoQlO into seminal fluids, sperm preparations and semen extenders has also proved equivocal, because the products used were insoluble in water, leading to precipitation of the CoQlO in the resultant fluid and ‘clumping’ of sperm around insoluble particles.

Also, the suspension of CoQlO in alcohol, chloroform or dimethyl sulfoxide (DMSO) to increase its solubility is not satisfactory either and may be damaging to the resultant embryo.

CoQlO of unspecified form has been used in studies with the aim of improving the quality of animal sperm including a study by Yanhu Wang et al (Animal Science Journal March, 2022: Coenzyme Q10 improves the quality of sheep sperm stored at room temperature by mitigating oxidative stress). CoQlO was found to improve the quality of ram semen by alleviating oxidative stress, 50 pmol/L CoQlO was found to be the optimum concentration.

A study by Monique de Albuquerque Lagares et al (J. Equine Vet Sci. May 2020; 88:102964) has shown that the addition of CoQlO, of unspecified form, exerts an antioxidant effect on frozen equine sperm. No improvement was found in sperm motility and kinetic characteristics. However, the investigators only used semen in which the progressive motility was >50% from a small number of horses (n=5) and this may have greatly reduced the chances of demonstrating an effect of reduction of ROS activity on progressive motility.

There is a need for a means of potentiating the effect of CoQlO, due to its high potency as an antioxidant, and, thereby, reducing oxidative stress, in enhancing the viability of egg and sperm cells for use in assisted reproduction of human and non-human animals in a reproducible and reliable manner.

In particular, there is a need for enhancing sperm motility, especially if this enhancement can be induced rapidly, thereby avoiding the need for a male (human or animal) to embark on a lengthy oral supplementation program in the hope of achieving the same effect.

Summary of the Invention

Thus, the invention provides an in vitro method for enhancing the effective functionality of a reproductive cell, which method comprises supplementing the endogenous ubiquinol of the cell by contacting a medium containing said reproductive cell with an aqueous solution in which ubiquinol is uniformly dispersed.

The method according to the invention can directly enhance the effective functionality of egg and sperm, by providing ubiquinol in a water- dispersible/dispersive form, a form in which it is active in an aqueous environment rather than rapidly precipitating as is the case with widely available forms of either form of CoQlO due to their marked hydrophobicity. As used herein, the terms water-dispersible and water-dispersive are used interchangeably.

We have found that using CoQlO, specifically its reduced form, ubiquinol, in a water-dispersible form, results in increased progressive mobility of sperm, for example in rams, whether cooled or frozen, and in sub-fertile stallions, as hereinafter described.

The method according to the invention, which is based on the use of water-dispersible ubiquinol, results in a reduction in oxidative stress, with the attendant advantages. In addition to the water-dispersible ubiquinol providing an antioxidant effect, it is likely that it also enhances the ability of sperm to generate ATP.

The method according to the invention has application in the assisted reproduction of human and non-human animals, including enhancing the fertility of animals used for food production for humans and other animals, by increasing litter size and/or conceptions/ejaculate and decreasing the period between parturition and the next/subsequent conception, and, thereby, overall livestock production rates stemming from artificial or assisted reproductive technologies.

The method according to the invention has application in the growing demand for improved animal efficiency and productivity, including the demand for animal protein, by increasing reproductive efficiency in all livestock species. Thus, the method according to the invention will have application in meeting the need for sustainable food production.

A further application of the method according to the invention facilitates the adoption of sexed semen in developing markets.

The method according to the invention could also have application in protecting endangered species by enhancing the fertility of such species.

In a preferred embodiment of the invention the reproductive cell is a sperm cell.

Seminal fluid can be contacted with the solution of ubiquinol, if desired.

Preferably, the concentration of ubiquinol used is in the range 30pg/ml to 150pg/ml, more especially of the order of 40pg/ml, when a low dose is required or a concentration of 75pg/ml, when a high dose is required.

Preferably, the solution of ubiquinol contains a suitable emulsifier and a thickener, to ensure water-dispersibility and stability of the ubiquinol after its addition to water.

Further, preferably, the emulsifier is starch or a modified starch such as starch octenylsuccinate or a salt thereof, at a ratio of at least 0.5 (mass ratio in terms of solid content) with respect to ubiquinol.

Preferably, the thickener is a gum such as gum Arabic at a ratio of 0.125 to 1 (mass ratio in terms of solid content) with respect to ubiquinol. Most preferably, the solution of ubiquinol contains starch octenyl succinate or a salt thereof as an emulsifying agent, more particularly the sodium salt, and gum arabic.

A suitable source of water-dispersible ubiquinol is marketed by the Japanese company Petroeuroasia Co., Ltd, which is currently used as an additive in the human athletics soft-drinks industry.

Preferably, the solution is buffered to a pH of between 6.8 and 7.2, most especially by using a phosphate buffer or phosphate buffered saline.

According to one embodiment of the invention, the solution is added to a sperm preparation.

According to a further embodiment the solution is added to a semen extender.

A suitable extender for non-human animals is INRA 82 or INRA 96 for centrifuging and cooling; for freezing suitable extenders include Triladyl and E-Z-Freezing. A suitable extender or sperm preparation for humans is that marketed as Sperm Prep®.

In general, a semen extender is a liquid diluent which is added to semen to preserve its fertilising ability. It acts as a buffer to protect sperm cells from their own toxic by-products, as well as protecting the sperm from cold shock and osmotic shock, for example, during chilling and shipping processes. The ‘sperm prep’ or equivalent in accordance with the invention can be used for washing and preparing fresh sperm for introduction into a female reproductive tract or receptacle used for IVF or other assisted reproductive process.

According to one embodiment of the invention, the semen extender can be used to preserve or protect the functionality of cooled or frozen animal semen for transport and insemination of multiple females.

Thus, the water-dispersible ubiquinol can be added to a semen extender used in a process of cooling sperm.

Alternatively, the water-dispersible ubiquinol can be added to a semen extender used in a process of freezing sperm.

Furthermore, the water-dispersible ubiquinol can be added to a semen extender used in a process of thawing sperm, more specifically previously cryopreserved sperm.

Sperm cells have limited CoQlO stores.

The CoQlO storage sites are in the mitochondrion/ mitochondria of the mid-piece of the sperm cell, depending on the structure of the species of sperm being treated. Water-dispersible ubiquinol, in accordance with the invention, can enter the sperm cell more readily than hydrophobic forms of reduced and oxidised forms of CoQlO. Thus, adding water- dispersible ubiquinol to sperm preparations and semen extenders should reduce oxidative stress, based on results of studies using hydrophobic forms of CoQlO -ubiquinone. ROS are ubiquitous throughout any given IVF cycle and they pose a significant threat to an embryonic culture, as gametes and embryos are highly sensitive to oxidative stress outside of the body.

According to the invention, the sperm preparation / ‘sperm prep’ can be used to enhance the functionality of compromised semen for insertion into a female subject.

According to a further embodiment of the invention, the solution of ubiquinol is used as a replacement for seminal fluid. However, in general, seminal fluid is removed.

In the case of humans, the semen is centrifuged to separate the seminal fluid from the spermatozoa, which are then removed. The seminal fluid is replaced by the addition of the ‘sperm prep’ fluid and the resultant fluid is incubated until its introduction into the female reproductive tract or a device designed for the purposes of supporting in vitro fertilization of an ovum or ova.

According to one embodiment, the method according to the invention extends the time for which progressive motility of sperm is increased by up to 20%, more particularly by up to 30% and especially by up to 40% relative to untreated sperm.

Thus, preferably, the method according to the invention extends the time for which progressive motility of sperm is increased by up to 40% relative to untreated sperm, with the attendant advantages, as hereinafter described. Accordingly, the method according to the invention extends the time for which progressive motility of sperm is maintained at a significantly higher level than would otherwise be the case in the event the sperm were being treated with an extender that did not contain water dispersible ubiquinol.

According to another embodiment, the method according to the invention increases the total motility of the ubiquinol-treated sperm by up to 20%, more particularly by up to 30%, especially by up to 40% and most especially by up to 50% relative to untreated sperm.

Thus, preferably, the method according to the invention increases the total motility of the ubiquinol-treated sperm by up to 50% relative to untreated sperm, with the attendant advantages, as hereinafter described.

Accordingly, the method according to the invention maintains the total motility of the sperm at a significantly higher level than would otherwise be the case in the event the sperm were being treated with an extender that did not contain water dispersible ubiquinol.

According to one embodiment of the invention, the treated reproductive cells are harvested for use in assisted reproduction.

In the method according to the invention, the reproductive cell can be obtained from a human or non-human mammal.

The invention also provides ubiquinol in water-dispersible form for use in an in vitro method for enhancing the effective functionality of a reproductive cell. Preferably, the reproductive cell used is a sperm cell.

The invention also provides a sperm preparation or semen extender supplemented with a water-dispersible form of ubiquinol.

The ubiquinol for use in accordance with the invention can be supplied in light-protected/opaque Winchester bottles, typically as two bottle sizes, namely 10ml and 30ml for low dose 40pg/ml and high dose 75 pg/ml, respectively and must be protected from oxidation. The solution would be dispensed in 0.2 ml aliquots by dropper.

Brief Description of the Drawings In the accompanying figures:

Fig. 1 is a graph of % Progressive Motility versus Time (mins) for goat semen, the subject of Example 2;

Fig. 2 is a graph of % Change in Motility for Total Motility and Progressive Motility after 120 mins from lOmins in low motility ej aculates, the subj ect of Example 2 ;

Fig. 3 is a graph of Rapid Progressive Motility % versus Time (mins/h) for ram semen, the subject of Example 3;

Fig. 4 is a graph of Rapid Progressive Motility % versus Time (mins/h) for ram semen, the subject of Example 4; and Fig. 5 is a graph of % Progressive Motility versus Time (mins/h) for stallion semen, the subject of Example 5.

Modes for Carrying out the Invention

The invention will be further illustrated by the following Examples.

Example 1

CoQlO is not readily soluble in semen extenders. A number of studies have been carried out to increase the solubility of CoQlO in semen extenders, using a variety of solvents, for example ethanol (Nogueira, B.G., et al., Coenzyme Q10 and a-tocopherol prevent the lipid peroxidation of cooled equine semen. Reproduction in Domestic Animals, 2015; 50(6): p. 1003-1010), DMSO (Yousefian, L, et al., Attenuation of cryopreservation-induced oxidative stress by antioxidant: Impact of Coenzyme Q10 on the quality of post-thawed buck spermatozoa. Cryobiology, 2018; 81: p. 88-93) and a combination of 0.1% water and 0.08% chloroform (Gualtieri, R., et al., Treatment with zinc, D-aspartate, and coenzyme Q10 protects bull sperm against damage and improves their ability to support embryo development. Theriogenology, 2014; 82(4): p. 592-598) with no apparent deleterious effects on semen quality. In each of the above examples, CoQlO was purchased from the Sigma- Aldrich Co.

Ruiz, A. et al (Effects of coenzyme Q10 (CoQlO) on equine semen quality after cryopreservation. Clinical Theriogenology, 2018; 10(3): 311) have found that repetitive mixing of the freezing extender E-Z Freezing “LE” ®, Animal Reproduction Systems, Chino, CA, USA containing CoQlO, also purchased from Sigma-Aldrich Co., with a laboratory mixer and no CoQlO solvents resulted in rapid precipitation due to the insolubility of the CoQlO, when the mixing ceased. Post-thaw addition of this semen extender preparation plus CoQlO to equine semen had no effect on progressive or total motility. It is postulated that the limited CoQlO solubility in aqueous solutions and the tendency for precipitation is likely to influence the availability of CoQlO to sperm cells.

In each of the following Examples 2-5, a ‘complete’ ejaculate was studied. The actual volume varied between species and animals, and within animals. Thus, the specific volume is not stated. Computerised Assisted Semen Analysis (CASA SpermVision®, MOFA Global, Verona, WI, USA), which is a widely used methodology, regardless of species, was used in the case of the following Examples. Any CASA- based assessment involves placing a drop of the sample on a slide; all drops being of the same size/volume.

Example 2

Effect of water-soluble ubiquinol on sperm with low motility in male goats

40 ejaculates (4 or 5 ejaculates/goat) were collected from 9 male goats under carefully supervised conditions at the same premises. Of the 40 ejaculates, 10 ejaculates from 3 goats were classified as having low motility based on CASA assessment of progressive and total motility (<50%). All ejaculates from one goat (n=4) had extremely poor progressive motility (<30%) in all ejaculates and these remained very low at each measurement time regardless of treatment status, so the analytical results for this goat were removed from the study.

The remaining 6 ejaculates were treated in the following manner to determine the effect of water-dispersible ubiquinol on sperm motility.

Each ejaculate was then split following collection and a standard INRA 96 (IMV Technologies, France) ruminant semen extender (control) or the same extender + 150 pmol/L (130pg/ml) water-dispersible ubiquinol (ubi) (ShiroQE, PetroEuroAsia Co., Ltd., China) was added to each of the split semen samples. Semen was incubated at 37°C and its quality was evaluated 10, 60 and 120 mins, following collection. After 2 hours better total motility and progressive motility in absolute terms and with respect to % change were observed in the samples to which ubiquinol had been added to the semen_extender when compared to the 10- minute value and the results in the Control (C) samples, as follows:

(Total motility: 32.3 ± 6.3 (C), 30.4 ± 4.3 (ubi) at 10 min; 40.3 ± 15.4 (C), 44.7 ± 15.5 (ubi) at 120 min; % change after 120 min: 126.3 ± 57.5 (C), 151.8 ± 70.6 (ubi); Progressive motility: 25.4 ± 5.9 (C), 23.6 ± 4.8 (ubi) at 10 min; 30.7 ± 10.5 (C), 33.3 ± 10.2 (ubi) at 120 min; % change after 120 min: 126.2 ± 5.0 (C), 150.7 ± 77.6 (ubi)). Total and progressive motility were determined by CASA.

It was further noted that it took some time for the benefits of adding ubiquinol to be detectable; 60 minutes was not a sufficient time for this.

The results for Progressive Motility are shown in Figs. 1 and 2 Statistical examination- 1 -way repeated measures analysis of variance for effects of treatment, on progressive and total motility, and % change in the same after 60 and 120 minutes (min) when compared to 10 min is shown in Table 1.

Table 1 Variable lOmin 60min 120min 60minA% 120minA%

Total motility p=0.48 p=0.56 p=0.006 p=0.92 p=0.013

Progressive motility p=0.41 p=0.31 p=0.004 p=0.70 p=0.043

Example 3

Ram Cooled Semen Study The semen from 6 commercially active rams was collected and pooled on 6 different days over a period of about 2 weeks. The semen from each of the rams was pooled on each collection day. Each pooled sample from each day was divided into 6 aliquots or replicates and each aliquot was then treated as an individual sample with multiple straws from each replicate being preserved until used in the study.

INRA 96 (IMV Technologies, France) semen extender with the addition of 150 pmol/L (130pg/ml) water dispersible ubiquinol, as used in Example 2, (“treatment”) or without (“control”) was added to the pooled semen and cooled at 5°C for 48 h. Rapid progressive motility* was assessed 10 mins, 24h, 25h, 48h and 49h after cooling. Samples from each of the 6 replicates of the cooled semen were placed in a water bath (37°C) between the 24^th and 25^th hours, and the 48^th and 49^th hours. Results:

Rapid progressive motility was greater in samples to which INRA plus ubiquinol had been used as the semen extender, when compared to the results from samples to which just the INRA had been added (p = 0.035).

*Progressive motility assessments in rams are divided into rapid progressive motility and slow progressive motility categories due to the characteristics of ram semen. Such a classification is not used for the semen of most other domestic animals.

The results are shown in Table 2 and Fig. 3.

Table 2 Fig. 3 shows that semen treated with INRA +ubiquinol had a positive effect on rapid progressive motility (p < 0.05).

Example 4

Ram Frozen Semen Study

The semen from the same 6 commercially active rams that contributed to Example 3 was collected and pooled on 6 different days over a period of about 2 weeks. The semen from each of the rams was pooled on each collection day. The pooled samples were then divided into 6 aliquots or replicates and each aliquot was frozen in liquid nitrogen vapour.

Prior to being frozen in liquid nitrogen vapour, each semen replicate had either Triladyl or Triladyl plus 150pmol/L (130pg/ml) water dispersible ubiquinol, as used in Example 2, added to it. Straws from each day’s pooled frozen ejaculates were randomly selected and then thawed and incubated at 37 °C in a water bath for 2 hours and rapid progressive motility was evaluated by CASA as soon as it was thawed and 10, 60 and 120 mins after thawing. Data was analysed by 1-way repeated measures ANOVA with the collection number in the sequence of collections as a blocking factor. Semen treated with INRA + ubiquinol had a positive effect on rapid progressive motility (p < 0.05).

Results Overall, there was a significant beneficial effect of treating the semen with Triladyl plus ubiquinol (pO.OOOl). Although rapid progressive motility decreased over time after thawing, samples with extender containing water dispersible ubiquinol had greater rapid progressive motility regardless of the time of the measurement as shown in Table 3 and Fig. 4.

Table 3

Different letters in Table 3 for a given measurement time denotes statistical significance across treatment groups (p < 0.05).

Example 5

Stallion Study with Cooled Semen

Semen from 3 stallions known to have varying ranges of semen quality (poor, good, excellent) was collected on multiple occasions (range: 5-12 times).

The semen and extender were prepared as follows: The diluent used for fresh samples evaluation was INRA 96 (IMV Technologies, France), which contains a purified fraction of milk caseins, penicillin, gentamicin, and the fungicide Amphotericin B.

Water dispersible ubiquinol, as used in Example 2, was added to the base diluent at a concentration of 150pmol/L (130pg/ml) Semen was cooled at 5 °C during a 48h period.

An aliquot was analyzed 10 minutes after the ejaculates were diluted, at 24 hrs. and 48hrs. The aliquot was incubated at 37°C for 10 min. prior to analysis.

Evaluation of semen quality

Fresh and cooled semen evaluation were performed at Ohs, 24hs and 48hs after a 10 min incubation at 37°C from every collection. An aliquot was withdrawn from the stored extended semen. Motility was determined by CASA.

Results:

As reflected by Fig. 5, the inclusion of ubiquinol with the INRA extender significantly improved the progressive motility of the semen from the stallion with the poor semen quality (p = 0.013).

The motility of the semen from the other two stallions was not significantly improved from a statistical sense, probably due to overall quality of the semen from those two animals. However, from a purely numerical perspective, there did appear to be some benefit to the addition of the ubiquinol based on assessment of progressive motility.

When the progressive motility results for all samples from all 3 stallions were analysed, there was again significantly better motility associated with the addition of ubiquinol to the INRA (p=0.012). The results are shown in Table 4 and Fig. 5

Table 4

Fig. 5 shows that semen treated with INRA +ubiquinol had greater progressive motility at each measurement time (p < 0.05).

Claims

Claims: -

1. An in vitro method for enhancing the effective functionality of a reproductive cell, which method comprises supplementing the endogenous ubiquinol of the cell by contacting a medium containing said reproductive cell with an aqueous solution in which ubiquinol is uniformly dispersed.

2. A method according to Claim 1, wherein the reproductive cell is a sperm cell.

3. A method according to Claim 2, wherein seminal fluid is contacted with the solution of ubiquinol.

4. A method according to any preceding claim, wherein the solution of ubiquinol contains starch octenylsuccinate or a salt thereof as an emulsifying agent, and gum arabic.

5. A method according to Claim 4, wherein the solution is buffered to a pH of between 6.8 and 7.2.

6. A method according to any one of Claims 2-5, wherein the solution is added to a sperm preparation.

7. A method according to any one of Claims 2-5, wherein the solution is added to a semen extender.

8. A method according to Claim 7, wherein the solution is added to a semen extender used in a process of cooling sperm.

9. A method according to Claim 7, wherein the solution is added to a semen extender used in a process of freezing sperm.

10. A method according to Claim 7, wherein the solution is added to a semen extender used in a process of thawing frozen semen.

11. A method according to any one of Claims 2 -5, wherein the solution of ubiquinol is used as an additive to seminal fluid.

12. A method according to any one of Claims 2-11, which extends the time for which progressive motility of sperm is increased by up to 40% relative to untreated sperm.

13. A method according to any one of Claims 2-11, wherein the total motility of the ubiquinol-treated sperm is increased by up to 50% relative to untreated sperm.

14. A method according to any one of Claims 1-13, wherein the treated reproductive cells are harvested for use in assisted reproduction.

15. A method according to any preceding claim, wherein the reproductive cell is obtained from a human or non-human mammal.

16. Ubiquinol in water-dispersible form for use in an in vitro method for enhancing the effective functionality of a reproductive cell.

17. Use according to Claim 16, wherein the reproductive cell is a sperm cell.

18. A sperm preparation or semen extender supplemented with a water-dispersible form of ubiquinol.