WO2004091570A1

WO2004091570A1 - Method of treatment

Info

Publication number: WO2004091570A1
Application number: PCT/NZ2004/000075
Authority: WO
Inventors: Craig Robert Bunt; Shane Burggraaf; Colin Roger Ogle; Michael John Rathbone; William Stanley Thompson; Billy Day
Original assignee: Interag
Priority date: 2003-04-17
Filing date: 2004-04-19
Publication date: 2004-10-28

Abstract

The present invention relates to a method of applying a fertility treatment to an animal characterised by the steps of altering the blood plasma level of a substance in the animal with respect to the endogenous blood plasma level of the substance, for a period of time sufficient to control oestrus, and maintaining the blood plasma level of the substance at a sufficiently high level to minimise compromising fertility.

Description

METHOD OF TREATMENT

TECHNICAL FIELD

This invention relates to a method of treatment.

In particular, this invention relates to a method of applying a fertility treatment for use in the controlled breeding of animals.

BACKGROUND ART

Considerable need has existed for many years for the availability of an effective means to control the occurrence of oestrus and ovulation in pigs. However, previous methods have been unable to maintain a sufficient progesterone plasma level to synchronise oestrus without compromising fertility.

The use of artificial insemination (Al) in swine in the U.S. during the past 5 to 10 years has increased from less than 5% to a level frequently estimated to be 35 to 40% at present, further highlighting the need to develop methods and devices to control the occurrence of oestrus in pigs.

In addition, most pig production units desire to use an "all in all out" system of operation, wherein all animals are moved between specialised units of the production cycle (such as breeding, farrowing, nursery units and so forth) at the same time. To have the animals grouped closely enough to allow this practice starts at the time of breeding and hence the requirement for control of oestrus and ovulation, particularly in gilts.

Further, other technologies can be implemented more effectively when animals in a group are of similar ages and/or reproductive status. For example, induced farrowing and cross-nursing of newborn pigs can be performed more effectively as can vaccinations, marketing, and so forth. A need also exists among researchers for an effective method to modify the oestrus cycle when using technologies requiring the collection of embryos, Al, embryo transfer, studies of follicular development, as well as related studies that desire closely grouped farrowing dates for nutrition experiments and so forth.

A wide range of substance delivery devices are used for the controlled breeding and reproductive management in a number of different animal species, including cattle, sheep and pigs.

These devices are typically designed to be retained within the body cavity for the slow release of an active substance over a period of time. In order to help retain the device within the body cavity, such devices typically feature various arms or wings to engage the walls of the cavity and thus attempt to retain the device against ejection.

Such devices are often used to synchronise oestrus in female animals prior to mating, where the devices are used intra-vaginally to release active substances such as progesterone. When the device is removed, plasma progesterone levels fall and the animals cycle in a controlled manner.

Typically these devices are constructed with a silicone matrix that has been impregnated with progesterone, allowing the slow release of progesterone for a given period of time. Release of progesterone from the matrix generally occurs at a specific dose rate. Whilst useful for animals requiring relatively low dose rates, the inability of the matrix to readily change dosages or deliver a higher dose has been a disadvantage.

In some animal species, the progesterone dose rate is critical for both synchronising oestrus and maintaining fertility. If plasma progesterone levels fall below critical levels during treatment, oestrus synchronisation may still occur but follicle integrity may be compromised thereby reducing conception rates and fertility. Some animal species such as pigs have been shown to require much higher levels of plasma progesterone for oestrus synchronisation, requiring dosage rates often not deliverable from impregnated matrix devices. Further, it has proved difficult to maintain high plasma levels of exogenous progesterone in pigs, likely due to down- regulation of endogenous progesterone levels and the metabolism and short half- life of progesterone in the body.

The physiology of the pig has also proved difficult to design intra-vaginal devices for that are capable of being retained for the length of time required for the delivery of the active substance.

In this regard, US 6,444,224 discloses a porcine intra-vaginal device featuring improved retention characteristics to ensure retention of the device against spontaneous ejection or removal by another animal.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicant reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents .form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process. It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided a method of applying a fertility treatment to an animal

characterised by the steps of

altering the blood plasma level of a substance in the animal with respect to the endogenous blood plasma level of the substance, for a period of time sufficient to control oestrus, and

maintaining the blood plasma level of the substance at a sufficiently high level to minimise compromising fertility.

According to another aspect of the present invention there is provided a method of applying a fertility treatment to an animal

characterised by the step of

delivering sufficient progesterone to maintain a progesterone blood plasma level of at least substantially 8 ng/ml, over a period of time sufficient to control oestrus and to minimise compromising fertility.

In preferred embodiments of the present invention the fertility treatment may be applied to pigs. However, it should also be appreciated that the present invention could find application in other animal species, including humans, and as such this should not be seen as a limitation. For ease of reference, the animal to which the invention is applied will hereinafter be referred to as a pig. The term "fertility treatment" should be taken to mean a treatment for use in controlled breeding and reproductive management.

In preferred embodiments the fertility treatment may be applied to animals prior to mating for the synchronisation of oestrus.

The term "blood plasma level" should be taken to mean the level of circulating substance in the blood of an animal.

The term "substance" should be taken to mean any substance capable of eliciting an effect on the controlled breeding and reproductive management in animals, including but not limited to proteins, hormones, antibodies and/or mimetics of these. For example; progesterone, flugesterone acetate, cloprostenol, dinoprost, oestradiol 17β, oestradiol benzoate, FSH, GnRH, and melatonin could all be used.

In preferred embodiments of the present invention the active substance is progesterone and/or a progesterone mimetic. For ease of reference throughout the specification the active substance will herein be referred to as progesterone, though this should not be seen as a limitation.

The term "period of time" should be taken to mean the length of time at which elevated progesterone levels are maintained to bring about the synchronisation of oestrus without compromising fertility.

It has previously been demonstrated by the applicant that in pigs, a minimum of 10 days is required to synchronise oestrus. Time periods of less than 10 days did not allow the synchronisation of oestrus, and also often resulted in the decrease of fertility due to follicle cysts.

In preferred embodiments the period of time over which the fertility treatment is applied is 10-15 days. It is anticipated that treatments lasting longer than 15 days could also be used, though this would likely not provide additional advantages and thus would be uneconomic.

It is anticipated that the minimum plasma level for any given substance will differ depending on the requirements of each animal species. Further, due to the nature of animal variability within each species it is anticipated that a device which provides a given plasma substance level in one animal may achieve a different plasma substance level in another animal.

In preferred embodiments the blood plasma progesterone level required to control oestrus and minimise compromising fertility in pigs equates to at least substantially 8 mg/ml as measured in an overiectomised animal, over at least a 10 day period.

Compromised fertility is evident by reduced conception rates and is often typified by the formation of ovarian and follicular cysts.

Progesterone may be delivered by any method which will induce a sustained increase in circulating progesterone such as by intravenous infusion, by a controlled release device, or so forth. However, this should not be seen as limiting and a number of methods known in the art could be used.

It is known that in order to effect the control of oestrus, a sustained increase in the circulating level of progesterone and/or progesterone mimetics is required, prior to the reduction to normal or low levels after infusion. Merely providing daily injections of exogenous progesterone would not be sufficient to alter the response of an animal. Progesterone is known to have a half-life in cows of approximately 15 minutes. Therefore, the applicant anticipate that if serial doses were delivered, such doses could be delivered no less than every approximately 7-8 minutes in order to be effective. Accordingly, methods that deliver a continuous, controlled release of progesterone are required to effect a sustained increase in progesterone levels.

In another preferred embodiment of the present invention the increase in the circulating level of progesterone may be brought about by the incorporation into the animal's genome of an inducible recombinant nucleotide sequence encoding biologically active progesterone or a recombinant nucleotide sequence encoding a molecule which enhances endogenous progesterone activity.

For example, the progesterone gene sequence could be inserted into an inducible gene cassette under the control of a suitable promoter, such as a promoter that expresses in all cell types (constitutive expression), or the progesterone promoter and/or a suitable enhancer sequence to drive transcription thereof. This cassette would also preferably contain 3' flanking DNA that could stabilise the mRNA and may contain downstream regulatory sequences.

This DNA cassette could be introduced into the genome of an animal by microinjection of the DNA into pronuclei of eggs (described by L'Huillier et al., 1996) which are subsequently transferred back to recipient animals and allowed to develop to term. This technique for the production of transgenic animals is described by Hogan et al. (1994).

Another way to produce transgenic animals involves transfection of cells in culture that are derived from an embryo, or foetal or adult tissues followed by nuclear transfer and embryo transfer to recipient animals. Alternatively, the gene cassette may be bound to mammalian sperm and delivered to the egg via in vitro or in vivo fertilisation to produce a non-human transgenic animal.

In transgenic animals, the induced increase in circulating levels of progesterone may be reduced after a desired period of time by switching off the inducible gene cassette, allowing the onset of oestrus. In another preferred embodiment of the present invention the alteration of progesterone levels may be achieved by the administration of antibodies capable of affecting the response of progesterone receptors to circulating progesterone levels.

These antibodies may have stimulatory or inhibitory effects on progesterone receptors, act as progesterone mimetics, or may bind to circulating progesterone or progesterone mimetic molecules to keep these in circulation for longer, prolonging the physiological response.

Implanted devices utilising osmotic pumps have previously been used to deliver high levels of progesterone to animals. However, whilst such implants were able to synchronise the onset of the oestrus cycle, poor fertility often resulted due to the formation of follicle cysts.

Likewise, oral doses suffer from the obvious limitations regarding the inability to accurately monitor the dose received by the animal and rate of application.

The applicant has discovered the location of the delivery device in the pig, in addition to the level and length of treatment, to be important.

In preferred embodiments the fertility treatment will be applied to a pig using an intra-vaginal substance delivery device.

Maintaining a high level of plasma progesterone for a sufficient period of time by delivering progesterone using an intra-vaginal device has been shown by the applicant to be beneficial for controlling oestrus and maintaining or enhancing fertility.

According to another aspect of the present invention there is provided a method of applying a fertility treatment to an animal substantially as described above

characterised by the step of using at least two substance delivery devices sequentially, to maintain a progesterone blood plasma level at a sufficiently high level for a period of time required to control oestrus and minimise compromising fertility.

Synthetic compounds that are orally effective and administered at prescribed doses on a daily basis have previously been used, though growing concern about contamination in meat has meant the use of a natural compound such as progesterone is favoured.

Previous attempts to use intra-vaginal sponges and other devices have been largely ineffective in pigs due to lack of a high rate of retention and also due to other animals extracting the devices by pulling on external attachments used to remove the insert at the end of the treatment period.

The applicant has found that prior art devices such as those described by US Pat. No. 6,444,224 provide sufficient progesterone to synchronise the onset of the oestrus. However, the device is unable to deliver additional progesterone to maintain a higher progesterone plasma level over the treatment period greater than those indicated in US Pat. No. 6,444,224.

This is likely due to a number of factors, including slower progesterone diffusion rates from the matrix over time, down-regulation of endogenous progesterone levels and the metabolism and short half-life of progesterone in the animal. The applicant has found that removing the original device on day 10, followed by insertion of a new device for an additional four days, to be of particular benefit to minimise compromising fertility.

In other embodiments a single delivery device may be used that is able to deliver sufficient progesterone to maintain a progesterone blood plasma level of at least substantially 8 ng/ml, over a period of time sufficient to control oestrus and minimise compromising fertility, substantially as described above.

The applicant has discovered that in order to control oestrus and minimise compromising fertility, sufficient progesterone must be delivered to maintain a progesterone blood plasma level of at least substantially 8 ng/ml, over the treatment period.

By maintaining a progesterone plasma level of at least substantially 8 ng/ml for 10 to 15 days, the applicant was able to demonstrate a significant effect on the control of oestrus in pigs, in addition to the average number of piglets per litter that were previously not obtainable using existing treatment methods.

Further, examination of ovaries from test animals revealed these animals had not developed ovarian or follicular cysts.

The ability to control the onset of oestrus in pigs without compromising fertility has important implications for the controlled breeding and reproductive management of pigs. Such methods will enable higher conception rates to be achieved from controlled breeding techniques such as Al, embryo collection and transfer, lowering the costs of such techniques.

Further, the increased use of Al allows animals to be grouped closely together in age, allowing many treatments such as vaccinations to be performed more efficiently, thereby lowering labour costs. The ability of the present invention to effectively modify the oestrus cycle also allows researchers to better conduct statistically valid studies into aspects of animal biology such as follicular development, to further increase conception and pregnancy rates.

According to another aspect of the present invention there is provided a delivery device for applying a fertility treatment to an animal according to the method substantially as described above.

Preferably the delivery device includes at least one of the following parameters,

i) a progesterone impregnated matrix, or

ii) a solid or semi-solid formulation of progesterone for delivery by a syringe like device.

One preferred embodiment of the delivery device is an easily moulded form capable of being moulded initially with a nylon spine about which there is then at least partially moulded or fabricated an encasement of progesterone impregnated silicone rubber, as described by US Pat. No. 6,444,224.

While prior art devices such as those described by US Pat. No. 6,444,224 provide sufficient progesterone to synchronise the onset of the oestrus, they may result in low or compromised fertility.

However, removing the first device on day 10, followed by insertion of a second device for an additional four days, has been found by the applicant to be of particular benefit and minimises compromising fertility.

Other preferred embodiments of the delivery device may utilise a device as described by US Pat No. 5,741 ,275 powered by an electrochemical cell as described by U.S. Pat. No. 5,245,565, which have been shown by the applicant to provide sufficient progesterone over the treatment period. The present invention thus provides methods for the control of oestrus which maintain a blood plasma progesterone level at sufficiently high levels to minimise compromising fertility, allowing better control over many aspects of controlled breeding and reproductive management in pigs.

To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as hereinbefore defined.

While the invention has been described in relation to the control of oestrus, it is anticipated that it could also be used for other purposes, such as the induction of parturition or so forth and as such this should not be viewed as a limitation.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following, description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 Shows a side elevation of one preferred embodiment of a device for use in the present invention; and

Figure 2 Shows average plasma progesterone concentration following insertion of a device as shown in figure 1 ; and

Figure 3 Shows a side elevation of another preferred embodiment of a device for use in the present invention; and

Figure 4 Shows average plasma progesterone concentration following insertion of a device as shown in figure 3. DETAILED DESCRIPTION OF THE INVENTION

Figures 1 and 3 show a porcine intra-vaginal device of a shape and size adapted to be positionable in the vaginal tract across the hymenal ring of a target animal (eg; a gilt) to extend to both sides of the hymenal ring of the animal, substantially as described in US 6,444,224.

One preferred embodiment of a device for use in the present invention as shown by Figure 1 utilises a syringe like device for the delivery of a liquid or semisolid progesterone formulation as described by US Pat No. 5,741 ,275 powered by an electrochemical cell as described by U.S. Pat. No. 5,245,565.

A progesterone formulation consisting of progesterone USP (20.8%w/w), Tween 40 (0.1 %w/w), hydroxypropyl methylcellulose NF (3%w/w) and water (76.1%w/w) is used.

Figure 2 shows the average animal plasma progesterone concentration during a fertility experiment following insertion of the device shown by Figure 1. As depicted, the average plasma progesterone is able to be maintained at substantially 8 ng/ml or above for a period of 14 days. This is followed by a rapid fall in plasma progesterone following removal of the device after day 14.

Table 1 is a summary of fertility treatments following administration for 14 days of a device as depicted in Figures 1 and 3.

Table 1. Summary of fertility results following administration for 14 days of a device as depicted in Figure. 1.

Characteristic Treatment

Number of gilts 11 Number of gilts excluded from analysis 1

Day of oestrus cycle at treatment (range) 6-18

Post Treatment

Oestrus by day 7 (%) 100

Average interval to estrus (days) 3.9

Ovarian Morphology

Number of corpora hi tea (mean) 17.9 Gilts with mature follicles (%) 0

Gilts with 1,2 or 3 cysts (%) 0

Gilts with over 4 cysts (%) 0

Fertility at day 4-7

Conception rate (%) 100

Number of embryos recovered (mean) 12.8

The results from Table 1 show that the treatment using the device had a significant effect on animals coming on heat or average number of piglets born. Examination of the ovaries of the test animals revealed that the test animals had not developed cysts on their ovaries.

Another preferred embodiment of a device for use in the present invention is shown by Figure 3. The delivery device is an easily moulded form capable of being moulded initially with a nylon spine about which there is then at least partially moulded or fabricated an encasement of progesterone impregnated silicone rubber, as described by US Pat. No. 6,444,224. The active ingredient of the device is micronised USP natural progesterone. Device potency is determined by the percentage of active ingredient present in the inactive silicone elastomer.

The progesterone is mixed into each of two liquid silicone parts prior to the silicone being introduced to the machine for moulding. A suitable two liquid system is that of Dow Corning Co marketed as Q74840 parts A and B.

At the moulding stage the two parts of the liquid silicone are pumped under pressure of approximately 100 bar from pails into the injection chambers of an injection moulding machine. Upon injection, the two parts of silicone are simultaneously forced through a static mixer before flowing into an electrically heated mould.

The nylon spine is inserted into the mould prior to the silicone being injected. The mould has a die surface temperature of typically 150 degrees to 190 degrees Celsius, but preferably never exceeding 200 degrees Celsius. The mould is kept clamped shut under approximately 30 tonnes of static pressure while the silicone cures. At the indicated temperature and pressure, the liquid silicone takes approximately 50 seconds to cure into a rubber.

Following curing, the finished product is removed from the mould and cooled before packaging.

Examples of materials that might be use for the spine and/or the initial part of the body include nylon and polyester.

Examples of materials that might be used include to carry the progesterone are silicone, polycaprolactone, EVA, starch derivatives and polyesters.

However, devices disclosed by US Pat. No. 6,444,224 has been shown previously be unable to deliver sufficient progesterone to maintain a high progesterone plasma level over the treatment period required to synchronise oestrus without compromising fertility.

As shown by Figure 4, the levels of plasma progesterone delivered by the Figure 3 device fall below substantially 8 ng/ml from approximately day 10 onwards.

With reference to Table 2, when the device of Figure 3 is not replaced and instead is utilised for 14 days, a negative effect on fertility is observed. This negative effect is believed by the applicant to arise from a failure to maintain sufficiently high blood progesterone levels in some individuals.

To investigate the period of time required for the administration of exogenous progesterone, devices shown by Figure 3 were either administered for 14 days, or were removed and replaced with a fresh device on days 10 or 12.

When the device of Figure 3 was removed on days 10 or 12 and replaced with a fresh device, this negative effect on fertility was greatly reduced, as can be seen with reference to Table 2.

Table 2. Summary of fertility results following administration, of a device as depicted in Figure. 2, for 14 days including replacement by a fresh device on days 10 or 12. w.

14 Day CIDR Treatment

No Day 12 Day 10

Characteristic Replacement Replacement Replacement

Number of gilts 65 55 56

Number of gilts excluded from analysis 5** 4*

Estrus by day 7 (%) 98% 100% 100%

Average interval to estrus (days) 3.9 4.9 4.4

Number of corpora lutea (mean) 13 16.8 18.6

Gilts with mature follicles (%) 18.0% 8.5% 5.9%

Gilts with 1 ,2 or 3 cysts (%) 10.7% 19.1 % 13.7%

Gilts with over 4 cysts (%) 24.6% 17.0% 5.8%

Conception rate (%) 60.7% 89.6% 84.6%

Number of embryos recovered (mean) 8.6 13.8 14.6

•CIDR Expelled = 75% Other = 25%: ** CIDR Expelled = 0% Other = 100% *** CIDR Expelled = 25% Other = 75%

Table 2 is a summary of fertility treatments following administration of the device shown by Figure 3 and as described by US 6,444,224.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims

WHAT WE CLAIM IS;

1. A method of applying a fertility treatment to an animal

characterised by the steps of

2. A method as claimed in claim 1 wherein the substance is progesterone.

3. A method as claimed in claim 1 or claim 2 wherein the animal is a pig.

4. A method as claimed in claim 3 including the further step of delivering sufficient progesterone to maintain a progesterone blood plasma level of at least substantially 8 ng/ml, over a period of time sufficient to control oestrus and to minimise compromising fertility.

5. A method as claimed in any one of claims 1 to 4 wherein the fertility treatment is applied to an animal prior to mating for the synchronisation of oestrus.

6. A method as claimed in any one of claims 1 to 5 wherein the period of time is a minimum of 10 days.

7. A method as claimed in any one of claims 1 to 6 wherein the period of time is 10-15 days.

8. A method as claimed in any one of claims 1 to 7 wherein the alteration of the blood plasma level of the substance is brought about by a sustained increase in the circulating level of substance and/or mimetics of said substance, followed by a reduction to normal or low levels.

9. A method as claimed in claim 8 wherein the sustained increase is brought about by intravenous infusion.

10. A method as claimed in claim 8 wherein the sustained increase is brought about by way of a controlled release device.

11. A method as claimed in claim 8 wherein the sustained increase is brought about by the incorporation into the animal's genome of an inducible recombinant nucleotide sequence encoding biologically active progesterone.

12. A method as claimed in claim 8 wherein the sustained increase is brought about by the incorporation into the animal's genome of a recombinant nucleotide sequence encoding a molecule which enhances endogenous progesterone activity.

13. A method as claimed in claim 11 or claim 12 wherein the recombinant nucleotide sequence contains 3' flanking DNA to stabilise an mRNA.

14. A method as claimed in any one of claims 11 to 13 wherein the recombinant nucleotide sequence contains downstream regulatory sequences.

15. A method as claimed in any one of claims 11 to 14 wherein the recombinant nucleotide sequence is inserted into an inducible gene cassette under the control of a suitable promoter and/or enhancer sequence.

16. A method as claimed in claim 15 wherein the promoter and/or enhancer sequence drives the transcription of the recombinant nucleotide sequence.

17. A method as claimed in claim 15 or claim 16 wherein the reduction in the circulating level of substance and/or mimetics of said substance is brought about by switching off the inducible gene cassette, allowing the onset of oestrus.

18. A method as claimed in claim 8 wherein the sustained increase is brought about by the administration of antibodies.

19. A method as claimed in any one of claims 1 to 18 wherein the fertility treatment will be applied to a pig using an intra-vaginal substance delivery device.

20. A method as claimed in 19 wherein at least two substance delivery devices are used sequentially, to maintain a substance blood plasma level at a sufficiently high level for a period of time required to control oestrus and to minimise compromising fertility.

21. A method as claimed in claim 20 wherein the first delivery device is removed on day 10, followed by insertion of a second delivery device for an additional four days.

22. A delivery device for applying a fertility treatment according to the method of claims 1 to 21.

23. A method of applying a fertility treatment to an animal substantially as herein described with reference to and as illustrated by the accompanying description and drawings.