WO2001097802A1

WO2001097802A1 - Supplement to enhance fertility

Info

Publication number: WO2001097802A1
Application number: PCT/GB2001/002640
Authority: WO
Inventors: Ray Clifford Noble
Original assignee: Jsr Clover Ltd
Priority date: 2000-06-17
Filing date: 2001-06-18
Publication date: 2001-12-27
Also published as: GB2363331A; GB2363331A8; AU2001274232A1; GB2363331B; GB0014758D0; EP1292295A1

Abstract

A supplement comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA for use in the promotion of fertility and reproductive output in animals and humans.

Description

SUPPLEMENT TO ENHANCE FERTILITY

This invention is concerned with the provision of a supplement by oral or other means and in particular one that exhibits properties for promoting and enhancing fertility and reproductive output in mammals such as livestock and particularly pigs.

In commercial pig breeding it is desirable to optimise male fertility and also increase the reproductive output of the females in order to maximise the breeder's commercial interests and subsequent financial return. In order to address these issues investigations were carried out into the function and composition, at a cellular level of male pig fertility and also the effect on the female reproductive output.

It has already been well documented in the prior art that spermatozoa and associated seminal plasma contain lipids (fat) which have been found to be unique in their composition and fatty acid constituents when compared with almost all other tissues within the male animal body. Generally all tissues have a requirement to maintain significant levels of essential polyunsaturated fatty acids. These usually exist as C18 components, linoleic and alpha-linolenic acids, which are distributed between phospholipid and neutral lipid fractions. Such acids are also accompanied by varying, but usually very much lower, concentrations of longer chain acids of higher polyunsaturation. These long chain acids can either be derived directly through nutrition or indirectly through synthesis by processes within the tissues themselves involving interrelated desaturation (introduction of more double bonds) and chain elongation (addition of more carbon atoms) of both linoleic and alpha-linolenic acid. As a result a selection of C20 and C22 polyunsaturates can arise from linoleic acid and these are known collectively as n-6 or omega-6 polyunsaturates. Likewise, by a similar process, a series of C20 and C22 polyunsaturates arise from alpha-linolenic acid and are collectively known as n-3 or omega-3 polyunsaturates.

In major animal tissues, the brain being a notable exception, the C20 and C22 polyunsaturates of the n-6 and n-3 series almost invariably together account for a maximum of about 6 per cent of the total fatty acids present.

Surprisingly, however, it has been found that in both the spermatozoa and seminal plasma of fresh ejaculates, the levels are extraordinarily high and in several instances account for up to 60-70 per cent of the total fatty acids present. Furthermore, the lipid present in spermatozoa and associated seminal

^' plasma consists almost entirely of phospholipid in which polyunsaturation is directed during spermatogenesis to specific moieties and molecular configurations. There is also discrete localisation of the lipids in distinct cellular domains. Unlike other major farm animal species and in fact humans, the male pig has a substantial level of polyunsaturates present within its spermatozoa and seminal fluid which is characteristically dominated by docosahexaenoic acid (DHA) of which its chain length is 22 carbon atoms with 6 double bonds in n-3 configuration and docosapentaenoic acid (DPA) of which its chain length is 22 carbon atoms with 5 double bonds in n-6 configuration. Together these two acids account for some 65 per cent of the total fatty acids present within the spermatozoa and seminal fluid with a distribution in each of between 25 and 35 per cent for each.

The present invention was developed to address the issues of maximising financial gain from commercial pig breeding by way of optimising male fertility whilst additionally increasing female reproductive output.

Preliminary observations indicated that in male pigs a reduction in spermatozoa levels of DHA and a concomitant increase in DPA was associated with reduced semen quality. As a result in depth investigations were carried out as to how an increase of DHA would affect fertility if at all and if it could be further enhanced by introducing the additional DHA alone or in combination with other components.

The object of the present invention was to produce a supplement for easy use and which was adapted to maximise the fertility and reproductive output in pigs.

According to the present invention there is provided a supplement for the promotion of fertility and reproductive output in animals comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA.

According to the present invention there is provided a supplement for the promotion of fertility and reproductive output in humans comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA.

Advantageously gamma-linolenic acid (GLA) may be added thereto.

According to the present invention there is provided a supplement for the promotion of fertility and reproductive output in animals comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant, selenium as the major cationic enzyme co-factor to protect the DHA and gamma-linolenic acid (GLA). According to the present invention there is provided a supplement for the promotion of fertility and reproductive output in humans comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant, selenium as the major cationic enzyme co-factor to protect the DHA and gamma-linolenic acid (GLA).

Preferably the DHA is present in the admixture at a level of 63-90g/kg.

Preferably the GLA is present in the admixture at a level of 4-10g/kg and advantageously is introduced in the form of oil from the Evening Primrose or Starflower.

Preferably the antioxidant is alpha-tocopherol and present in the admixture at a minimum of lOOOmg/kg.

Moreover the selenium is present in the admixture at a minimum 2.75mg/kg.

Optionally stearadonic acid, an n-3 C18 polyunsaturated acid, may be added to the admixture.

The admixture can be introduced into the animal by way of a suitable carrier for ingestion by the animal such as feedstuff s or by gelatin bolus. Moreover, the fatty acids, antioxidants and cationic cofactors can be supplied intramuscularly, intraperitonealy, intravenously or subcutaneously for slow release and appropriate tissue targeting as chelates, chemical complexes and adjuncts.

Additionally the properties of the supplement promote increased fertility in both the male and female animals. The properties of the supplement increased significantly a wide range of qualitative and quantitative biochemical/ physiological parameters of the semen and its spermatozoa resulting in the promotion of the_t fertilising capacity of the ejaculate. Additionally the application to gilts/ sows of such semen from boars supplemented with the admixture resulted in a significant elevation to female reproductive output and efficiency with appropriate substantial economic returns in terms of progeny.

Preferably the admixture is fed at 250g/male pig/ day.

The invention will now be described in more detail below with reference to the examples and Figures detailed below.

Figure 1 - graph showing the spermatozoa concentration in semen from control and supplemented boars according to Group 2 in Table A; Figure 2 - graph showing mean spermatozoa concentration and total sperm number in the semen from control and supplemented boars according to Group 2 in Table A;

Figure 3 - graph showing total born and born alive progeny to gilts inseminated with semen from control and supplemented boars according to Group 2 in Table A;

Figure 4 - graph showing farrowing rate and fecundity for gilts inseminated with semen from control and supplemented boars according to Group 2 in Table A; and

Figure 5 - graph showing mean obtainable artificial inseminations per ejaculate for control and supplemented boars according to Group 2 in Table A.

Examples

Investigations were performed on breeding male pigs maintained solely under standard conditions appropriate to commercial semen production for artificial insemination and thus well known in the art. The investigations are divided into 3 basic groups in which semen output, their major characteristics and fertilising capacities were compared between boars that received (daily) a standard breeder diet (basal diet) only with boars that received both the standard diet together with 0.25kg of an oral supplement daily.

In all the investigations the experimental groups consisted of matched (age, weight etc.) boars and gilts, from the same genetic stock and maintained under the conditions referred to above. The diets were fed for 16 weeks with individual semen samples taken at day 0 and 4 week intervals thereafter for appropriate qualitative and quantitative investigations.

In all three groups control boars were fed daily with a standard breeder (basal) diet supplying <5mg DHA and 250mg of the antioxidant alpha-tocopherol. Table A

Supply of DHA, GLA, a-Toco herol and selenium per day to the control and experimental boars

The major fatty acid components of the standard (basal) diet, the enriched DHA oil and GLA source (Evening Primrose Oil) are given below in Table 1.

Table 1.

Fatty acid compositions (wt. per cent of total fatty acids) of the basal diet and the supplemental high DHA and GLA oils.

Following collection from the animals in the control and in Groups 1-3, the spermatozoa were harvested from the semen by gentle centrifugation as per standard commercial procedures well known in the art. The physiological and chemical measurements were then undertaken immediately and the parameters measured according to standard procedures well known in the art of semen production and spermatozoa physiology and biochemistry. Those parameters measured were as follows:

1. ej aculate volume ; 2. spermatozoa concentration by both spectrophotometric optical density and haemocytometry;

3. spermatozoa motiliry by microscopy and Cellsoft^® Computer Assisted Analysis; and

4. percentage of live, dead and malformed spermatozoa by differential cell staining.

Total lipid content was extracted from the spermatozoa by an established procedure involving an exhaustive extraction with organic solvents. Lipid separation and quantification was then subsequently performed by thin layer chromatography. Fatty acid analysis of the lipids was performed again by established procedures involving transesterification and appropriate application to capillary gas liquid chromatography.

The fertilising capacity of the semen was measured in vivo by artificial insemination of gilts as per commercial practice and conception rates, fecundity (piglet numbers, born alive /dead etc.) recorded on an individual boar and gilt basis.

Statistical comparison of data was performed using Student's t-test, analysis of variance and correlations as appropriate.

RESULTS

Group 1

Table 2 shows the long chain polyunsaturated fatty acid compositional changes that occurred within the spermatozoa as a result of the supplemental addition outlined in this group. It can be seen that there was a substantial elevation in the level of DHA at the expense of a proportional reduction in that of DPA. In the supplemented group the level of DPA:DHA in the spermatozoa (in the supplemented boars at 16 weeks of feeding) was 13.8:45.5(wt per cent of total fatty acids) versus 25.1:32.8 in those fed the standard diet.

As result there was a considerable positive enhancement of the n-3/n-6 fatty acid ratio. Although observable positive trends on a range of spermatozoa features e.g. concentration, proportion of live/dead cells, structural morphology, increased motility, were observed across the experiments, the biological scatter was such that statistical significance was not reached. Substantially increasing the availability of alpha-tocopherol failed to promote further the polyunsaturate changes within the spermatozoa.

Table 2.

Fatty acid composition (wt. per cent of total fatty acids) of the phospholipid fraction of the spermatozoa from the control and DHA supplemented boars at 16 weeks of feeding.

Group 2

Table 3.

Comparative function parameters of the spermatozoa in the semen from control and supplemented (DHA/ alpha- tocopherol / selenium) boars at week 16.

TVS: Total viable sperm:

VSC: Viable sperm concentration ^a = significantly different at a minimum of(P<0.05)

As can be seen from Table 3 supplemental addition of DHA, alpha- tocopherol and selenium resulted in a similar effect upon spermatozoa polyunsaturated fatty acid composition and n-3/n-6 ratio to that for Group 1. However, the effect upon all the measured spermatozoa production and functional parameters (see Table 3 and Figures 1 and 2) was highly positive with statistical significant achieved throughout. As can be seen from Figures 3 and 4 effects upon progeny outcomes in terms of piglets born alive to the gilts, farrowing rates and fecundities were also all highly significant. Figure 5 shows the significant effect of the increased spermatozoa output in terms of commercially obtainable artificial inseminations per ejaculate.

When the semen from the supplemented boars is stored at 16°C under the usual conditions appropriate to pig artificial insemination, a significant increase in spermatozoa viability was observed compared to control boars in terms of: a) the maintenance of the proportion of spermatozoa with high motility; and b) a reduction in the number of damaged cells observable from the 3^rd day up to the 9^th day following semen collection.

These features were associated throughout the period with the maintenance with the spermatozoa of the enhanced level of DHA.

Group 3

The additional inclusion of GLA in the prefeed supplement of this group enhanced further both the concentration and total spermatozoa output per ejaculate (11.4 versus 6.4 x 10⁸ per ml and 85 versus 62 x 10⁹ per ejaculate respectively). In all the experiments significant changes in the respective levels of the polyunsaturates were observed from the 4^th week of supplementation onwards, peaking at the 8^th week. Investigation of the phospholipid composition showed an increase in DHA and reduction in DPA was associated in particular with the phosphatidyl ethanolamine component. There were highly positive correlations (P<0.006) between the levels of DHA within the spermatozoa and the spermatozoa characteristics for the data from groups 1 and 2.

It should be noted that although the examples show the use of alpha- tocopherol, other antioxidants can also be used, such as plant extracts. Also other fatty acids such as Stearadonic acid (n-3) also have been found to enhance fertility in accordance with this invention.

Amongst major farm animal species the pig is unique in displaying for one reason or another a high proportion of DPA at the expense of DHA within the lipid of the spermatozoa.

One of the advantages of the present invention is that now the promotion of male fertility in the pig under present intensive commercial production systems can be promoted through enhanced supplemental DHA availability to maximise the DHA level within the spermatozoa and its metabolic involvement in spermatozoa output, quality and functionality.

The present invention clearly demonstrates the ability to increase substantially the level of DHA at the expense of DPA in the spermatozoa of the modern commercial male breeding pig through supplemental dietary means. However for any effectiveness of such fatty acid changes on spermatozoa functional parameters to be achieved, it was quite clearly shown that there was a requirement for the supplemental polyunsaturates to be accompanied by additional dietary selenium which was accompanied by an enhanced glutathione peroxidase activity (group 2). Under these circumstances a wide selection of significant effects on spermatozoa reproductive features were observed. A further improvement on the spermatozoa features was effected by supplemental inclusion also of GLA (group 3). This combination of polyunsaturates and cationic cofactor in the presence of enhanced levels of alpha-tocopherol produced significant responses across a full range of quantifiable physiological features of major importance to spermatozoa performance when compared with spermatozoa obtained from pigs maintained on normal modern breeding dietary situations. Such benefits were not only manifested in spermatozoa parameters but also notably and most uniquely in the response by gilts /sows following artificial insemination of semen from treated boars, in terms of conception, born alive and overall reproductive fecundity which in turn demonstrated major efficiency benefits to both reproductive output and economic return. The achieved manipulation of fatty- acid-oxidant composition of boar spermatozoa through a supply of exogenous substrate and accompanied by an appropriate anti-oxidant portfolio, provided a unique and significant alteration to the spermatozoa composition. That influenced and promoted spermatozoa survival during residence within the uterine -oviduct environment of the gilt/ sow during oestrus and thereafter the time window prior to ovulation. Combined with this beneficial effect is a strong association between spermatozoa membrane lipid infrastructure with the process of capacitation and fusion mechanism of oocyte attachment, leading to increased fertilisation rates.

The effects observed under a strict modern pig breeding situation at both male and female levels has therefore significant implications for contemporary commercial pig production and breeding in terms of investment returns and savings for the pig producer.

Although it is envisaged that the supplement herein described be used for livestock, in particular pigs, it is to be understood that due to the well known similarity between pigs and human biochemistry and physiology , the supplement would also find application in maximisation of human fertility and reproductive output.

Therefore the advantages of the present invention include the fact that the addition of a supplement based on a unique formulation of a high DHA containing oil and alpha-tocopherol influenced significantly the C22 polyunsaturate acid proportions within the spermatozoa and the proportion of DHA was considerably increased at the sole expense of DPA. Under these conditions there was a trend for a positive effect upon a range of spermatozoa features associated with fertilising capacity. With the addition of selenium to the supplement the effect of the fatty acid changes on the full range of spermatozoa fertilising features were all highly significant. Further, administering the semen from boars receiving the DHA/ a-T/ selenium supplement to gilts via artificial insemination effected a significant increase in fertilisation and progeny output as judged on a range of commercial parameters. Moreover, incorporation of GLA into the supplement further enhanced the semen quality of the boar. Also the admixture provided by this application significantly extends, under semen storage conditions appropriate to artificial insemination, the maintenance within the spermatozoa of several features important to fertilising capacity. Finally, based on modern commercial pig production and breeding practice the supplementation resulted in a highly significant increase in financial return.

Claims

1. A supplement comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA for use in the promotion of fertility and reproductive output in animals.

2. A supplement comprising an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA for use in the promotion of fertility and reproductive output in humans.

3. A supplement according to claims 1 or 2 wherein the admixture further comprises gamma-linolenic acid (GLA).

4. A supplement according to claims 1 or 3 when dependent on 1 wherein the DHA present in the admixture is in the range of 63-90g/kg.

5. A supplement according to claim 3 when dependent on 1 or 4 wherein the GLA present in the admixture is in the range of 4-10g/kg.

6. A supplement according to claim 5 wherein the GLA is in the form of oil from the Evening Primrose.

7. A supplement according to claim 5 wherein the GLA is in the form of oil from the Starflower.

8. A supplement according to claims 1, 3 when dependent on 1 or 4-7 wherein the amount of antioxidant present in the admixture is at least lOOOmg/kg.

9. A supplement according to claim 8 wherein the antioxidant is alpha- tocopherol.

10. A supplement according to claims 1, 3 when dependent on 1 or 4-9 wherein the amount of selenium present in the admixture is at least 2.75mg/kg.

11. A supplement according to any one of the preceding claims wherein the supplement is contained in a carrier vehicle for introduction to the recipient.

12. A supplement according to claim 11 wherein the vehicle comprises a bolus.

13. A supplement according to claim 11 wherein the vehicle comprises an injectable formulation.

14. A supplement according to claims 1, 3 when dependent on 1 or 4-13 wherein the admixture is introduced to the recipient at an amount of 250g/male pig/ day orally.

15. A supplement as substantially herein described.

16. Use of an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA in preparation of a supplement for promoting fertility and reproductive output in animals.

17. Use of an admixture of an enriched triacylglycerol source of Docosahexaenoic acid (DHA) accompanied by at least one antioxidant and selenium as the major cationic enzyme co-factor to protect the DHA in preparation of a supplement for promoting fertility and reproductive output in humans.

18. Use according to claims 16-17 wherein the supplement is a supplement of any one of claims 3-15.