CA2047723A1 - Compound for improving the ovary reactions and the ova and embryo production in domestic mammals in combination with biotechnological embryo transfer - Google Patents
Compound for improving the ovary reactions and the ova and embryo production in domestic mammals in combination with biotechnological embryo transferInfo
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- CA2047723A1 CA2047723A1 CA002047723A CA2047723A CA2047723A1 CA 2047723 A1 CA2047723 A1 CA 2047723A1 CA 002047723 A CA002047723 A CA 002047723A CA 2047723 A CA2047723 A CA 2047723A CA 2047723 A1 CA2047723 A1 CA 2047723A1
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- compound
- inhibin
- antibodies
- embryo
- ova
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Endocrinology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
The drug for improving the reaction of the ovary and the production of ova and embryos in domestic mammals in connection with biotechnological embryo transfer is characterized in that it consists of inhibin-neutralizing antibodies or of said antibodies in combination with hormones which promote superovulations, which are directed against animal and human, natural or recombinant inhibin or its subunits, fragments and derivatives, and can neutralize the biological activity of the inhibin produced in the gonads and thereby bring about, within one reproductive cycle, multiple follicle growth and multiple follicle ripening up to multiple ovulation by passive immunization, and by its alternative embodiment and by a process for producing it.
The drug for improving the reaction of the ovary and the production of ova and embryos in domestic mammals in connection with biotechnological embryo transfer is characterized in that it consists of inhibin-neutralizing antibodies or of said antibodies in combination with hormones which promote superovulations, which are directed against animal and human, natural or recombinant inhibin or its subunits, fragments and derivatives, and can neutralize the biological activity of the inhibin produced in the gonads and thereby bring about, within one reproductive cycle, multiple follicle growth and multiple follicle ripening up to multiple ovulation by passive immunization, and by its alternative embodiment and by a process for producing it.
Description
~326 7'~
COMPOUND FOR IMPROVING THE OVARY REACTIONS
AND THE OVA AND EMBRYO PRODUCTION IN DOMESTIC MAMMALS
IN COMBINATION WITH BIOTEC~NOLOGICAL EMBRYO TRANSFER
. . _ It is known that with regard to mammals which 5 within a single reproduction cycle as a rule produce in a natural manner only a single fertilized ova (monotocous animal species), to be able to cause an increase in the production of ova and, as a result therefrom, of embryos through the utilization of gonodotropenes; for example, ~ such as HCG, HMG, PMSG, FSH, LH and/or GnRH, which controls the release of gonadotropenes from the hypophysis.
Consequently~ it is thereby possible to successively produce a series of ovas which are 15 fertilizable, and to recover these as embryos subsequent to their fertilization, and to transfer these to recipient animals. These stimulation methods for the achieving of superovulations has the disadvantage that this can be achieved only through an excess dosing with 20 gonadotropic hormones. Due to the endogenic counter-regulation against multiple ovulations, especially with monotocous domestic mammals, unsatisfying ovary reactions result from such known stimulation methods especially with respect to the extense interacting and internal and 25 individual variability, as well as the therefrom resulting number o~ ~ualitatively high-grade embryos. A
significant multiple usage of oogenises and oozytes which ,.
~ - , : . .
COMPOUND FOR IMPROVING THE OVARY REACTIONS
AND THE OVA AND EMBRYO PRODUCTION IN DOMESTIC MAMMALS
IN COMBINATION WITH BIOTEC~NOLOGICAL EMBRYO TRANSFER
. . _ It is known that with regard to mammals which 5 within a single reproduction cycle as a rule produce in a natural manner only a single fertilized ova (monotocous animal species), to be able to cause an increase in the production of ova and, as a result therefrom, of embryos through the utilization of gonodotropenes; for example, ~ such as HCG, HMG, PMSG, FSH, LH and/or GnRH, which controls the release of gonadotropenes from the hypophysis.
Consequently~ it is thereby possible to successively produce a series of ovas which are 15 fertilizable, and to recover these as embryos subsequent to their fertilization, and to transfer these to recipient animals. These stimulation methods for the achieving of superovulations has the disadvantage that this can be achieved only through an excess dosing with 20 gonadotropic hormones. Due to the endogenic counter-regulation against multiple ovulations, especially with monotocous domestic mammals, unsatisfying ovary reactions result from such known stimulation methods especially with respect to the extense interacting and internal and 25 individual variability, as well as the therefrom resulting number o~ ~ualitatively high-grade embryos. A
significant multiple usage of oogenises and oozytes which ,.
~ - , : . .
2~L~2~
is necessitated through an overdosing, especially with gonadotropenes, leads to a reduction in the potential live or vital production yield of a donor animal.
Such an intense intervention into the endogenic 5 and endocrinous regulation of Eolliculogeneses, follicle ripening and into the ovulation processes, lead to temporary and even to permanent derivative damages to the reproductive yield of donor animals.
These disadvantages which are encountered in 10 the state-of-the-technology are avoided by the compound pursuant to the invention.
To the extent that in accordance with the state-of-the-art there are employed materials which ac~
as inhibin, this utilization is implemented as an active ~5 immunization or for the examination of the effects on the physiological reproduction parameters. In accordance with the extent that in the state-of-the-art, there are employed materials which are efffective as inhibin-neutralizing antibodies, this utilization is effected for 20 diagnostic proof and for the attaining of a profertility effect for individual, female mammals.
Fertility signifies the capability for reproduction of an individual and the capacity of the individual to produce a certain number of live offspring 25 durlng each reproductive period.
:.
3477~
A utilization for the causing of a superovulation is, in contrast therewith, not a fertility-propagating measure.
The subject matter of the present invention is 5 to provide a compound for an improvement in the ovary reactions, and in the ova and embryo production for domestic mammals in combination with biotechnological embryo transfer or transplation.
It is an object of the compound pursuant to the invention that through a controlled intervention into the central endogenic endocrinous regulation of the ovulation rate-control systems, to initiate the superovulation and, as a result thereof, develop a more universal method for superovulation stimulation for different species of 15 domestic mammals.
Furthermore, it is another object o~ the invention to achieve more constant overreactions and a higher proportion of qualitatively high-grade embryos which are capable of being transferred or transplanted, 20 and to thereby improve the economics of the embryo transfer for domestic mammals.
The solution of the object pursuant to the invention is set forth in the characterizing portion of Claim l. The compound pursuant to the invention is 25 specified in preferred alternative embodiments in the subclaims.
~ ~ ~7~'~3 1 The compound pursuant to the invention is characterized in that it consists of inhibin-neutralizing antibodies which are directed against animal species-differing and human, naturally occurring or recombinant 5 tsynthetic) inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological activity of the inhibin which is formed in gonads and thereby cause through immunization, within a single reproduction cycle, multiple follicle growth and multiple 10 follicle ripening up to multiple ovulation (superovulation) for domestic mammals. Furthermore, the compound pursuant to the invention is characterized by another, independent property. This is characterized in that this consists of inhibin-neutralizing antib~ ies in 15 combination with multiple ovulations 5supero~lation~-propagating hormones, preferably with gonadotropenes and/or with GmRH (gonadotropin-releasing hormone, and/or with antiestrogenes in the form of synthetic active materials or antibodies, preferably glomiphene or 20 tamoxifene, and/or with anti-androgenes in the form of synthetic active materials or antibodies, preferably against androsteron, testosteron, dihydrotestosteron, which is directed against animal species-differing, and human, naturally encountered or recombinant (synthetic) 25 inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological 77~
.. . .
1 activity of the inhibin Eormed in gonads and thereby, within a single reproductive cycle, produces through immunization multiple follicle growth and multiple follicle ripening up to superovulation.
The compound pursuant to the invention is characterized alternatively and in a preferred embodiment in it is constituted from specific polyclonal or monoclonal antibodies (antiserum) which neu~ralize the biological activity of inhibin in living organisms.
The compound pursuant to the invention is also alternatively and preferably characterized in that it is constituted from antibodies acting against naturally encountered or recombinant inhibins or their subunits, or their fragments, or their derivatives, and whose complete 5 molecule represents a glycoprotein which preferably consists as heterodimers from two different subunits ~a and ~), bonded through di-sulfide bridges, and whose molecular weight consists of 5 to 120 kDalton ~kDa) and which selectively suppress the FSH-secretion.
The compound pursuant to the invention is also alternatively and preferably characterized in that it is applied while in a liquid phase, preferably through intra~enous and~or subcutaneous and/or through intramuscular application.
The compound pursuant to the invention is also alternatively and preferably characterized through a process for its utilization. This process is characterized in that the application of the compound is implemented as multiple injections at specified time intervals, preferably of 12 or 24 hours, over a number 5 of successively following days, preferably on 3.5 to 5 days.
This process is also alternatively and preferably characterized in that the application is implemented during the middle luteal phase, pre~erably on 10 the 7th through the 11th day of the cycle.
Furthermore, the process of the invention is also characterized in that the application is carried out with equal or reducing dosages, with presently two equal successively following quantities.
l~ The following authors describe the characterization of an inhibin (antigen) for the species of animal designated as cattle. Robertson, D.M, et al.;
Molecular and Cellular Endocrinology, 44 ~1986) pages 271 - 277.
Forage, R.G., et al.; Proc~ National Academy of Science, USA (1986) pages 3091 - 3095.
Described is the isolating and characterization through cloning and sequence analysis of DNA, which code for the subunits of inhibin from bovine follicle fluid 25 (bFF).
~ . ~ . . : :
~7~3 Y~
1 Produced was the 31 kDa- form of the inhibin through the introduction oE a pH- ~.75 precipitation step into the described purifying method for the 58 kDa-inhibin from bFF. Consequently, the former is a clearage 5 product from 58 kDa-inhibin.
The compound pursuant to the invention sets forth that to be understood under antibodies are such which neutralize and thereby render ineffective the biological activity of the inhibins which are produced in 0 the gonads of the animal.
Miyamoto K., et al., in Biochemlcal and Biophysical Research Communications, Vol. 136, No. 3 (1986), pages 1103 - 1109, describe inhibin-antibodies for the species of animals designated as cattle. These 15 are monoclonal antibodies employed against the bFF 32 kDa-inhibin from bovine follicle fluid.
Monoclonal antibodies against bFF 32 kDa-inhibin are known from the utilization of the hybridizing techni~ue in mice (origin Balb/c).
~ A certain antibody, Type 256 H, recognizes specifically the bovine 20 kDa (~-subunit) and the 13 kDa (~ subunit) of bovine 32 kDa-inhibin. In comparison with other antibodies this evidences a strict specificity for the bovine 20 kDa-subunit.
The compound of the invention pursuant to Claim 1 is advantageous in the e~uine species of animals, due - ,, . .. :
-: - . , :
2C1 ~77~3 1 to the lacking in the response of this species of animal to gonadotropene preparates.
The compound pursuant to Claim 2 must be reflected upon due to the ovary reaction in most domestic 5 mammals.
The utilization of the compound of the invention pursuant to Claim 2; for example, is described as follows:
There is verified the effectiveness of the 10 combination of inhibin-neutralizing antibodies (antiserum) in combination with hormones which propagate superovulations (gonadotropin). Available from an agricultural operation are 15 head of cattle of the type "Deutsche Schwarzbunte" with a cross-breading of 15 approximately 75% "Holstein-Frisian" within the age groups of 17.5 to 25 months and in the weight classes of about 350 to 410 kg.
These cows were presynchronized with injections twice of each respectively of 500 ~g chloprosterol, sold 20 under the trademark "Estrumate"; a commercial product of the Coopers Tierarzneimittel GmbH, 3006 Burgwedel, Germany.
The injections were administered at an interval of 11 days. Thereafter, the cows were divided into two 25 groups in accordance with the random principle.
_~_ ~ 3 1 ~t the beginning of the treatment, the cows were at the 9th day of the cycle.
The cattle from Group I, the control group, with n = 8 cows, were administered a total dosage of 5 23.8 mg NIH-FSH unit-e~uivalent (National Institute of Health-FSH) at 12 hour intervals over 3.5 days in a decreasing dosages with 4.2 / 4.2 / 3.5 / 3.5 / 2.8 2.8 / 2.8 mg FSH-equivalent. There were additionally included in each of the 6th and 7th injections 10 respectively 500 ~g chloprostenol for the initiation of the luteolysis. With regard to chloprostenol is water there is to be understood as water-soluble sodium salt.
The artificial insemination was carried out 36, 48, 60, 72 hours after the initiation of the luteolysis with 15 deep-frozen sperm from a single bull.
The cattle from Group II, with n = 7 cows, in addition to the above-described treatment, and concurrently with the FSH injections, were intravenously injected each with 20 ml of an antiserum pooled from four 20 sheep against inhibin S-antiserum.
For the production of this serum, four sheep rams were actively immunized with a purified preparate of inhibin from bovine follicle fluid, bFF, which was obtained in a slaughterhouse for cattle.
The cattle from whose ovaries there was obtained the follicle fluid, were previously treated with : . . .
2~7~3 j;
1 PMSG for increasing the inhibin concentration of inhibin in the bFF.
The rams were initially each basically immunized with respectively 2.5 mg protein o~ a puri~ied 5 Inhibin-preparate, dissolved in 2.0 ml of an 0.9% NaCl solution and with 2.0 ml complete Freund's adjuvant.
The booster administrations were effected on the 20th and 50th days subsequent to the basic immunization with, respectively, 2.0 and 1.8 mg protein, 10 dissolved in 2.0 ml o~ an 0.9% NaCl solution and with 2.0 ml of incomplete Freund's adjuvant.
The serum recovery was carried out on the 7th and 10th days subse~uent to the second boostering. The antiserum from the four rams were pooled with the same ~5 volumetric parts and ~ractionated, 150 ml, and stored at -20C.
Achieved were the ~ollowing results:
Treatment: Ova/Embryos Components n Cl +/- total Folltropin 23.8 mg 8 10.5 5.88 7.9 Folltropin 23.8 mg + 7 14.1 3 .29 9.6 S-Antibodies against inhibin Treatment: Ova/Embroys 7~2~3 ~
1 Components ~/- fertilized +/- % transferable +/- %
Folltropin 23.8 mg 4.76 5.1 3.27 66 3.8 3.06 48 Folltropin ~3.8 mg+ ?.64 8.6 2.44 90 8.3 2.21 86 S-Antibodies against inhibin The numerical values are indicated with the median deviations (on-1) as average values, which result 1O from the arithmetic median of the sum of the individual values of the animals.
Folltropin is a gonadotropin preparate obtained from hyperphasis cells of slaughtered pigs with a low lh constituent. There were employed: Falltropin, a 15 commercial product of Vetrepharm Inc., London, Ontario, Canada. Selled in Germany by: Praemix GmbH, Mannheim, Germany.
In the for~going table there signifies:
n = number of experimental animals, Gl = yellow body, determined through rectal palpation, as a feature of the number of implemented ovulations, Total = number of all obtained ovas, unfertilized and the embryos, % = numerical values referring to "total", 1 Superovulation-propagating hormone =
Folltropin, commerclal product.
The treatment with Folltropin in Group I or the control group, displays an average obtention of 7.9 5 embroys and ova for each individual animal. The proportion of fertilized embryos consisted of 66% (5.l) and the proportion of implantaLble or transferable embryos of 48% t3.8). The compound pursuant to the invention with the combination of antibodies and gonadotropenes 10 leads to an unexpected and significant increase in the obtention of ova and embryos to 9.6 and the proportion of fertilized embryos up to 90% (8.6) and the transferable embryos up to 86% ~8.3).
When an average rate of pregnancies of 60% was 5 employed as the basis resulting from the embryo transfer, then from 3.8 transferable embryos, Group I, in practice there could be expected an average of 2.3 pregnancies for the animals.
The 8.3 embryos which were capable of transfer 20 by means of the compound of the invention, in contrast therewith led unexpectedly to an increase to 5.0 expected pregnancies.
The compound pursuant to the invention thus leads in a surprising manner to a doubling in the number 25 of offspring of a donor animal in contrast with ~he state-of-the~art.
: ` :
~'7~
~ ~urther practical experiment was carried out under similar conditions in another operation with cattle.
Available ware 7 cattle of the same species in 5 the age classes of about 16 to 19 months and in the weight classes of about 320 to 360 kg. The synchronization, as well as the further treatment of the cattle was carried out as in the first experiment.
The cattle in Group I, control group, with 10 n = 3, in a deviation from Experiment 1 were treated with a total dosage of 16.9 mg NIH-FSH standard-equivalent, Folltropin. The individual dosages consisted of 3.15 /
is necessitated through an overdosing, especially with gonadotropenes, leads to a reduction in the potential live or vital production yield of a donor animal.
Such an intense intervention into the endogenic 5 and endocrinous regulation of Eolliculogeneses, follicle ripening and into the ovulation processes, lead to temporary and even to permanent derivative damages to the reproductive yield of donor animals.
These disadvantages which are encountered in 10 the state-of-the-technology are avoided by the compound pursuant to the invention.
To the extent that in accordance with the state-of-the-art there are employed materials which ac~
as inhibin, this utilization is implemented as an active ~5 immunization or for the examination of the effects on the physiological reproduction parameters. In accordance with the extent that in the state-of-the-art, there are employed materials which are efffective as inhibin-neutralizing antibodies, this utilization is effected for 20 diagnostic proof and for the attaining of a profertility effect for individual, female mammals.
Fertility signifies the capability for reproduction of an individual and the capacity of the individual to produce a certain number of live offspring 25 durlng each reproductive period.
:.
3477~
A utilization for the causing of a superovulation is, in contrast therewith, not a fertility-propagating measure.
The subject matter of the present invention is 5 to provide a compound for an improvement in the ovary reactions, and in the ova and embryo production for domestic mammals in combination with biotechnological embryo transfer or transplation.
It is an object of the compound pursuant to the invention that through a controlled intervention into the central endogenic endocrinous regulation of the ovulation rate-control systems, to initiate the superovulation and, as a result thereof, develop a more universal method for superovulation stimulation for different species of 15 domestic mammals.
Furthermore, it is another object o~ the invention to achieve more constant overreactions and a higher proportion of qualitatively high-grade embryos which are capable of being transferred or transplanted, 20 and to thereby improve the economics of the embryo transfer for domestic mammals.
The solution of the object pursuant to the invention is set forth in the characterizing portion of Claim l. The compound pursuant to the invention is 25 specified in preferred alternative embodiments in the subclaims.
~ ~ ~7~'~3 1 The compound pursuant to the invention is characterized in that it consists of inhibin-neutralizing antibodies which are directed against animal species-differing and human, naturally occurring or recombinant 5 tsynthetic) inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological activity of the inhibin which is formed in gonads and thereby cause through immunization, within a single reproduction cycle, multiple follicle growth and multiple 10 follicle ripening up to multiple ovulation (superovulation) for domestic mammals. Furthermore, the compound pursuant to the invention is characterized by another, independent property. This is characterized in that this consists of inhibin-neutralizing antib~ ies in 15 combination with multiple ovulations 5supero~lation~-propagating hormones, preferably with gonadotropenes and/or with GmRH (gonadotropin-releasing hormone, and/or with antiestrogenes in the form of synthetic active materials or antibodies, preferably glomiphene or 20 tamoxifene, and/or with anti-androgenes in the form of synthetic active materials or antibodies, preferably against androsteron, testosteron, dihydrotestosteron, which is directed against animal species-differing, and human, naturally encountered or recombinant (synthetic) 25 inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological 77~
.. . .
1 activity of the inhibin Eormed in gonads and thereby, within a single reproductive cycle, produces through immunization multiple follicle growth and multiple follicle ripening up to superovulation.
The compound pursuant to the invention is characterized alternatively and in a preferred embodiment in it is constituted from specific polyclonal or monoclonal antibodies (antiserum) which neu~ralize the biological activity of inhibin in living organisms.
The compound pursuant to the invention is also alternatively and preferably characterized in that it is constituted from antibodies acting against naturally encountered or recombinant inhibins or their subunits, or their fragments, or their derivatives, and whose complete 5 molecule represents a glycoprotein which preferably consists as heterodimers from two different subunits ~a and ~), bonded through di-sulfide bridges, and whose molecular weight consists of 5 to 120 kDalton ~kDa) and which selectively suppress the FSH-secretion.
The compound pursuant to the invention is also alternatively and preferably characterized in that it is applied while in a liquid phase, preferably through intra~enous and~or subcutaneous and/or through intramuscular application.
The compound pursuant to the invention is also alternatively and preferably characterized through a process for its utilization. This process is characterized in that the application of the compound is implemented as multiple injections at specified time intervals, preferably of 12 or 24 hours, over a number 5 of successively following days, preferably on 3.5 to 5 days.
This process is also alternatively and preferably characterized in that the application is implemented during the middle luteal phase, pre~erably on 10 the 7th through the 11th day of the cycle.
Furthermore, the process of the invention is also characterized in that the application is carried out with equal or reducing dosages, with presently two equal successively following quantities.
l~ The following authors describe the characterization of an inhibin (antigen) for the species of animal designated as cattle. Robertson, D.M, et al.;
Molecular and Cellular Endocrinology, 44 ~1986) pages 271 - 277.
Forage, R.G., et al.; Proc~ National Academy of Science, USA (1986) pages 3091 - 3095.
Described is the isolating and characterization through cloning and sequence analysis of DNA, which code for the subunits of inhibin from bovine follicle fluid 25 (bFF).
~ . ~ . . : :
~7~3 Y~
1 Produced was the 31 kDa- form of the inhibin through the introduction oE a pH- ~.75 precipitation step into the described purifying method for the 58 kDa-inhibin from bFF. Consequently, the former is a clearage 5 product from 58 kDa-inhibin.
The compound pursuant to the invention sets forth that to be understood under antibodies are such which neutralize and thereby render ineffective the biological activity of the inhibins which are produced in 0 the gonads of the animal.
Miyamoto K., et al., in Biochemlcal and Biophysical Research Communications, Vol. 136, No. 3 (1986), pages 1103 - 1109, describe inhibin-antibodies for the species of animals designated as cattle. These 15 are monoclonal antibodies employed against the bFF 32 kDa-inhibin from bovine follicle fluid.
Monoclonal antibodies against bFF 32 kDa-inhibin are known from the utilization of the hybridizing techni~ue in mice (origin Balb/c).
~ A certain antibody, Type 256 H, recognizes specifically the bovine 20 kDa (~-subunit) and the 13 kDa (~ subunit) of bovine 32 kDa-inhibin. In comparison with other antibodies this evidences a strict specificity for the bovine 20 kDa-subunit.
The compound of the invention pursuant to Claim 1 is advantageous in the e~uine species of animals, due - ,, . .. :
-: - . , :
2C1 ~77~3 1 to the lacking in the response of this species of animal to gonadotropene preparates.
The compound pursuant to Claim 2 must be reflected upon due to the ovary reaction in most domestic 5 mammals.
The utilization of the compound of the invention pursuant to Claim 2; for example, is described as follows:
There is verified the effectiveness of the 10 combination of inhibin-neutralizing antibodies (antiserum) in combination with hormones which propagate superovulations (gonadotropin). Available from an agricultural operation are 15 head of cattle of the type "Deutsche Schwarzbunte" with a cross-breading of 15 approximately 75% "Holstein-Frisian" within the age groups of 17.5 to 25 months and in the weight classes of about 350 to 410 kg.
These cows were presynchronized with injections twice of each respectively of 500 ~g chloprosterol, sold 20 under the trademark "Estrumate"; a commercial product of the Coopers Tierarzneimittel GmbH, 3006 Burgwedel, Germany.
The injections were administered at an interval of 11 days. Thereafter, the cows were divided into two 25 groups in accordance with the random principle.
_~_ ~ 3 1 ~t the beginning of the treatment, the cows were at the 9th day of the cycle.
The cattle from Group I, the control group, with n = 8 cows, were administered a total dosage of 5 23.8 mg NIH-FSH unit-e~uivalent (National Institute of Health-FSH) at 12 hour intervals over 3.5 days in a decreasing dosages with 4.2 / 4.2 / 3.5 / 3.5 / 2.8 2.8 / 2.8 mg FSH-equivalent. There were additionally included in each of the 6th and 7th injections 10 respectively 500 ~g chloprostenol for the initiation of the luteolysis. With regard to chloprostenol is water there is to be understood as water-soluble sodium salt.
The artificial insemination was carried out 36, 48, 60, 72 hours after the initiation of the luteolysis with 15 deep-frozen sperm from a single bull.
The cattle from Group II, with n = 7 cows, in addition to the above-described treatment, and concurrently with the FSH injections, were intravenously injected each with 20 ml of an antiserum pooled from four 20 sheep against inhibin S-antiserum.
For the production of this serum, four sheep rams were actively immunized with a purified preparate of inhibin from bovine follicle fluid, bFF, which was obtained in a slaughterhouse for cattle.
The cattle from whose ovaries there was obtained the follicle fluid, were previously treated with : . . .
2~7~3 j;
1 PMSG for increasing the inhibin concentration of inhibin in the bFF.
The rams were initially each basically immunized with respectively 2.5 mg protein o~ a puri~ied 5 Inhibin-preparate, dissolved in 2.0 ml of an 0.9% NaCl solution and with 2.0 ml complete Freund's adjuvant.
The booster administrations were effected on the 20th and 50th days subsequent to the basic immunization with, respectively, 2.0 and 1.8 mg protein, 10 dissolved in 2.0 ml o~ an 0.9% NaCl solution and with 2.0 ml of incomplete Freund's adjuvant.
The serum recovery was carried out on the 7th and 10th days subse~uent to the second boostering. The antiserum from the four rams were pooled with the same ~5 volumetric parts and ~ractionated, 150 ml, and stored at -20C.
Achieved were the ~ollowing results:
Treatment: Ova/Embryos Components n Cl +/- total Folltropin 23.8 mg 8 10.5 5.88 7.9 Folltropin 23.8 mg + 7 14.1 3 .29 9.6 S-Antibodies against inhibin Treatment: Ova/Embroys 7~2~3 ~
1 Components ~/- fertilized +/- % transferable +/- %
Folltropin 23.8 mg 4.76 5.1 3.27 66 3.8 3.06 48 Folltropin ~3.8 mg+ ?.64 8.6 2.44 90 8.3 2.21 86 S-Antibodies against inhibin The numerical values are indicated with the median deviations (on-1) as average values, which result 1O from the arithmetic median of the sum of the individual values of the animals.
Folltropin is a gonadotropin preparate obtained from hyperphasis cells of slaughtered pigs with a low lh constituent. There were employed: Falltropin, a 15 commercial product of Vetrepharm Inc., London, Ontario, Canada. Selled in Germany by: Praemix GmbH, Mannheim, Germany.
In the for~going table there signifies:
n = number of experimental animals, Gl = yellow body, determined through rectal palpation, as a feature of the number of implemented ovulations, Total = number of all obtained ovas, unfertilized and the embryos, % = numerical values referring to "total", 1 Superovulation-propagating hormone =
Folltropin, commerclal product.
The treatment with Folltropin in Group I or the control group, displays an average obtention of 7.9 5 embroys and ova for each individual animal. The proportion of fertilized embryos consisted of 66% (5.l) and the proportion of implantaLble or transferable embryos of 48% t3.8). The compound pursuant to the invention with the combination of antibodies and gonadotropenes 10 leads to an unexpected and significant increase in the obtention of ova and embryos to 9.6 and the proportion of fertilized embryos up to 90% (8.6) and the transferable embryos up to 86% ~8.3).
When an average rate of pregnancies of 60% was 5 employed as the basis resulting from the embryo transfer, then from 3.8 transferable embryos, Group I, in practice there could be expected an average of 2.3 pregnancies for the animals.
The 8.3 embryos which were capable of transfer 20 by means of the compound of the invention, in contrast therewith led unexpectedly to an increase to 5.0 expected pregnancies.
The compound pursuant to the invention thus leads in a surprising manner to a doubling in the number 25 of offspring of a donor animal in contrast with ~he state-of-the~art.
: ` :
~'7~
~ ~urther practical experiment was carried out under similar conditions in another operation with cattle.
Available ware 7 cattle of the same species in 5 the age classes of about 16 to 19 months and in the weight classes of about 320 to 360 kg. The synchronization, as well as the further treatment of the cattle was carried out as in the first experiment.
The cattle in Group I, control group, with 10 n = 3, in a deviation from Experiment 1 were treated with a total dosage of 16.9 mg NIH-FSH standard-equivalent, Folltropin. The individual dosages consisted of 3.15 /
3.15 / 2.45 / 2.45 /' 1.9 ~ 1.9 mg for the respective injections.
The cattle in the Group II, Experimental group with n = 4 cows, in addition to the treatment of the Control Group I, were concurrently intravenously injected against inhibin with the FSH injections each of 15 ml of an antiserum pooled from 6 rabbits.
The production of the rabbit antiserum, K-antiserum, against inhibin was carried out analogously with the method employed for the rams.
Z~77~3 . ;~
1 A~hieved were the following results:
Treatment: Ova/Embryos Components n Cl +/- total Folltropin 16.9 mg 3 14.7 4.95.3 Folltropin 16.9 mg+ 4 17.0 1.411.8 K-Antiserum against inhibin `~ ' .
~7~3 .j", .-Treatment: Ova/Embryos Components +/- fertiliæed +/- % transferable +/- %
Folltropin 16.9 mg 4.2 4.0 3.6 75 2.0 2.0 38 Folltropin 16.9 mg+ 2.2 10.3 1.3 87 6.5 1.9 63 K-Antiserum against inhibin The compound pursuant to the invention with the 10 combination of antibodies and gonadotropenes leads also in this experiment to a significant increase of the average obtained ova and embryos from 5.3 to 11.8, as well as to a significant increase in the proportion of fertili~ed embryos from 75% ~.O) to 87% (10.3) and in 15 the transferable embryos from 38% ~2.0) to 63% (6.5).
At an achievable pregnancy rate of 60~ from the embryo transfers, from 2.0 transferable embryos there could be expected an average of 1.2 pregnancies.
The utilization of the compound of the 20 invention, in contrast therewith, with 6.5 transferable embryos for each donor animal led to an increase of up to 3.9 expected pregnancies.
Thereby, the result of the first experiment was independently verified. This result is surprising.
The experiments were carried out by the inventor.
-. :
2~77~3 1 There could also be determined the following surprising result from the expleriments:
In both experimen-ts, the median deviation of the individual values from a median value (average value) 5 at higher absolute values of all investigated parameters in the groups treated with the compound pursuant to the invention were significantly lower than in the con-trol groups. An improvement in the stimulation is especially effected also through the more constant ovary reactions 10 from the compound of the invention.
The utilization of antibodies which neutralize inhibin is carried out pursuant to the state-of-the-art through single injections on the second or on the eighth day of the cycle.
This is verified by the following publications:
J. H. M. Wrathall, et al., Journal of Endocrinolog~ (1990) 124, 167 - 176. Refer to page 169, right-hand colu~n under 'IExperiment 2", lines 7 through 9: "On day 2".
~. E. Mann, et al., first column (1989) 123, pages 383 - 391. See page 385, left-hand column, under "Im~nunization experiment'`, line 15: "On day 8 of the luteal phase".
In contrast therewith, the compound pursuant to 25 the invention is applied as follows:
~4~'~2~3 There are administered a plurality of injections at defined time intervals, preferably in 12 or 24 hours over a number of succlessively following days, preferably between 3.5 and 4 days.
The treatment is carried out, in general, most optionally during the luteal phase, preferably from the 7th to the 11th cycle. During this interval, there appears on the ovaries a new wave of growing follicles.
A similar treatment scheme is applied during the embryo 0 transfer in the treatment with gonadotropin.
The compound pursuant to the invention affords the technical advantage of an increase production of qualitatively high-grade, transferable embryos for each scavenging of a donor animal at considerably more 15 constant ovary reactions.
The resultingly increased production of valuable offspring produces a significant increase in the economics of embryo transfer programs, (ET-Programs), especially with cattle.
The compound pursuant to the invention affords the further advantage that its employment is also possible in connection with such species of animals which could heretofore not be stimulated or only with a slight degree of success; especially, such as with mares.
.,, . , . ~-
The cattle in the Group II, Experimental group with n = 4 cows, in addition to the treatment of the Control Group I, were concurrently intravenously injected against inhibin with the FSH injections each of 15 ml of an antiserum pooled from 6 rabbits.
The production of the rabbit antiserum, K-antiserum, against inhibin was carried out analogously with the method employed for the rams.
Z~77~3 . ;~
1 A~hieved were the following results:
Treatment: Ova/Embryos Components n Cl +/- total Folltropin 16.9 mg 3 14.7 4.95.3 Folltropin 16.9 mg+ 4 17.0 1.411.8 K-Antiserum against inhibin `~ ' .
~7~3 .j", .-Treatment: Ova/Embryos Components +/- fertiliæed +/- % transferable +/- %
Folltropin 16.9 mg 4.2 4.0 3.6 75 2.0 2.0 38 Folltropin 16.9 mg+ 2.2 10.3 1.3 87 6.5 1.9 63 K-Antiserum against inhibin The compound pursuant to the invention with the 10 combination of antibodies and gonadotropenes leads also in this experiment to a significant increase of the average obtained ova and embryos from 5.3 to 11.8, as well as to a significant increase in the proportion of fertili~ed embryos from 75% ~.O) to 87% (10.3) and in 15 the transferable embryos from 38% ~2.0) to 63% (6.5).
At an achievable pregnancy rate of 60~ from the embryo transfers, from 2.0 transferable embryos there could be expected an average of 1.2 pregnancies.
The utilization of the compound of the 20 invention, in contrast therewith, with 6.5 transferable embryos for each donor animal led to an increase of up to 3.9 expected pregnancies.
Thereby, the result of the first experiment was independently verified. This result is surprising.
The experiments were carried out by the inventor.
-. :
2~77~3 1 There could also be determined the following surprising result from the expleriments:
In both experimen-ts, the median deviation of the individual values from a median value (average value) 5 at higher absolute values of all investigated parameters in the groups treated with the compound pursuant to the invention were significantly lower than in the con-trol groups. An improvement in the stimulation is especially effected also through the more constant ovary reactions 10 from the compound of the invention.
The utilization of antibodies which neutralize inhibin is carried out pursuant to the state-of-the-art through single injections on the second or on the eighth day of the cycle.
This is verified by the following publications:
J. H. M. Wrathall, et al., Journal of Endocrinolog~ (1990) 124, 167 - 176. Refer to page 169, right-hand colu~n under 'IExperiment 2", lines 7 through 9: "On day 2".
~. E. Mann, et al., first column (1989) 123, pages 383 - 391. See page 385, left-hand column, under "Im~nunization experiment'`, line 15: "On day 8 of the luteal phase".
In contrast therewith, the compound pursuant to 25 the invention is applied as follows:
~4~'~2~3 There are administered a plurality of injections at defined time intervals, preferably in 12 or 24 hours over a number of succlessively following days, preferably between 3.5 and 4 days.
The treatment is carried out, in general, most optionally during the luteal phase, preferably from the 7th to the 11th cycle. During this interval, there appears on the ovaries a new wave of growing follicles.
A similar treatment scheme is applied during the embryo 0 transfer in the treatment with gonadotropin.
The compound pursuant to the invention affords the technical advantage of an increase production of qualitatively high-grade, transferable embryos for each scavenging of a donor animal at considerably more 15 constant ovary reactions.
The resultingly increased production of valuable offspring produces a significant increase in the economics of embryo transfer programs, (ET-Programs), especially with cattle.
The compound pursuant to the invention affords the further advantage that its employment is also possible in connection with such species of animals which could heretofore not be stimulated or only with a slight degree of success; especially, such as with mares.
.,, . , . ~-
Claims (8)
1. A compound for the improvement of ovary reactions and for the ova and embryo production in domestic mammals in combination with biotechnological embryo transfer, characterized in that the compound consists of inhibin-neutralizing antibodies which are directed against-species differing and human, naturally occurring or recombinant (synthetic) inhibin or its subunits, or its fragments or its derivatives, and which neutralize the biological activity of the inhibin which is formed in gonads, and thereby, within a reproduction cycle, cause through immunization a multiple follicle growth and multiple follicle ripening up to multiple ovulation (superovulation) in domestic mammals.
2. A compound for the improvement of ovary reactions and for the ova and embryo production in domestic mammals in combination with biotechnological embryo transfers, in a modification of Claim 1, characterized in that the compound consists of inhibin-neutralizing antibodies in combination with hormones propagating multiple ovulations (superovulation) preferably with gonadotropenes and/or with GnRH
(gonadotropin-releasing hormone), and/or with antiestrogenes in the form of synthetic active materials or antibodies, preferably glomiphene, or tamoxifene, and/or with antiandrogenes in the form of synthetic active materials or antibodies, preferably against androtesteron, testosteron, dihydrotestosteron, which is directed against animal-species differing and human, naturally encountered or recombinant (synthetic) inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological activity of the inhibin formed in gonads and thereby, within a reproduction cycle, causes through immunization multiple follicle growth and multiple follicle ripening up to superovulation.
(gonadotropin-releasing hormone), and/or with antiestrogenes in the form of synthetic active materials or antibodies, preferably glomiphene, or tamoxifene, and/or with antiandrogenes in the form of synthetic active materials or antibodies, preferably against androtesteron, testosteron, dihydrotestosteron, which is directed against animal-species differing and human, naturally encountered or recombinant (synthetic) inhibin or its subunits, or its fragments or its derivatives, and which can neutralize the biological activity of the inhibin formed in gonads and thereby, within a reproduction cycle, causes through immunization multiple follicle growth and multiple follicle ripening up to superovulation.
3. A compound according to Claims 1 and 2, characterized in that the compound consists of specific polyclonal or monoclonal antibodies which neutralize the biological activity of inhibin in living organisms.
4. A compound as claimed in Claim 1 and 2, characterized in that the compound consists of antibodies acting against naturally encountered or recombinant inhibins or their subunits, or their fragments, or their derivatives, and whose complete molecule represents a glycoprotein, which consists preferably as a heterodimer from two different subunits (.alpha. and .beta.) which are bonded through disulfide bridges, and whose molecular weight is 5 to 120 kDalton (kDa) and which selectively suppress the FSH-secretion.
5. A compound as claimed in Claims 1 through 6, characterized in that the compound is administered in a liquid phase, preferably through intravenous and/or subcutaneous and/or intramuscular applications.
6. A process for the utilization of the compound according to Claims 1 through 5, characterized in that the administration is carried out in multiple injections at defined time intervals, preferably 12 or 24 hours over a number of successively following days, preferably of 3.5 to 5 days.
7. A process for the utilization of the compound according to Claim 6, characterized in that the administration is carried out during the middle luteal phase, preferably on the 7th to the 11th cycle.
8. A process for the utilization of the compound according to Claims 6 and 7, characterized in that the administration is carried out in equal or decreasing dosages with presently two equal successively following quantities.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4000327A DE4000327A1 (en) | 1990-01-08 | 1990-01-08 | MEDICINES FOR IMPROVING OVARREACTION AND EGG AND EMBRYONE PRODUCTION IN DOMESTIC MUSCLES |
| DEP4000327.2 | 1990-01-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2047723A1 true CA2047723A1 (en) | 1991-07-09 |
Family
ID=6397731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002047723A Abandoned CA2047723A1 (en) | 1990-01-08 | 1991-01-04 | Compound for improving the ovary reactions and the ova and embryo production in domestic mammals in combination with biotechnological embryo transfer |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0463138B1 (en) |
| AU (1) | AU649994B2 (en) |
| CA (1) | CA2047723A1 (en) |
| DE (3) | DE4000327A1 (en) |
| WO (1) | WO1991010449A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4000327A1 (en) * | 1990-01-08 | 1991-07-11 | Stefan Heiden | MEDICINES FOR IMPROVING OVARREACTION AND EGG AND EMBRYONE PRODUCTION IN DOMESTIC MUSCLES |
| JP2899534B2 (en) * | 1994-12-09 | 1999-06-02 | 全国農業協同組合連合会 | Method of inducing superovulation in cattle |
| EP4339206A1 (en) | 2021-12-15 | 2024-03-20 | Ningbo Sansheng Biological Technology Co., Ltd. | Antibody against inhibin and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1341617C (en) * | 1984-06-08 | 2011-06-28 | Henry George Burger | Inhibin isolated from ovarian follicular fluid |
| AU4437485A (en) * | 1984-06-08 | 1986-01-10 | Biotechnology Australia Proprietary Limited | Inhibin isolated from ovarian follicular fluid |
| US4864019A (en) * | 1985-11-08 | 1989-09-05 | The Salk Institute For Biological Studies | Antibodies to inhibin and conjugates produced therefrom |
| WO1988000208A1 (en) * | 1986-06-24 | 1988-01-14 | The Salk Institute For Biological Studies | Ovine inhibin |
| US4780451B1 (en) * | 1987-01-23 | 1995-04-04 | Asua International Inc | Composition and method for producing superovulation in cattle |
| DE4000327A1 (en) * | 1990-01-08 | 1991-07-11 | Stefan Heiden | MEDICINES FOR IMPROVING OVARREACTION AND EGG AND EMBRYONE PRODUCTION IN DOMESTIC MUSCLES |
-
1990
- 1990-01-08 DE DE4000327A patent/DE4000327A1/en active Granted
- 1990-01-08 DE DE4042408A patent/DE4042408C2/en not_active Expired - Fee Related
-
1991
- 1991-01-04 WO PCT/EP1991/000004 patent/WO1991010449A1/en active IP Right Grant
- 1991-01-04 AU AU70552/91A patent/AU649994B2/en not_active Ceased
- 1991-01-04 EP EP91902160A patent/EP0463138B1/en not_active Expired - Lifetime
- 1991-01-04 CA CA002047723A patent/CA2047723A1/en not_active Abandoned
- 1991-01-04 DE DE59101599T patent/DE59101599D1/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE4000327C2 (en) | 1992-07-02 |
| DE59101599D1 (en) | 1994-06-16 |
| EP0463138B1 (en) | 1994-05-11 |
| WO1991010449A1 (en) | 1991-07-25 |
| EP0463138A1 (en) | 1992-01-02 |
| AU649994B2 (en) | 1994-06-09 |
| DE4000327A1 (en) | 1991-07-11 |
| AU7055291A (en) | 1991-08-05 |
| DE4042408C2 (en) | 1995-04-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20040105 |