Increasing ovum collection in domestic cattle

Abstract

The present invention relates to methods and compositions for increasing ovum collection in domestic cows using follicle stimulating hormones.

Description

[0001] The present invention relates to methods and compositions for egg collection in bovine cows. The present invention also relates to methods and compositions for inducing, causing or stimulating superovulation in bovine cows. The present invention also relates to methods and compositions for increasing conception rates in bovine cows. The present invention can be used for artificial conception or in vitro fertilization. Background Art

[0002] The ability to improve the reproductive performance of non-human mammals such as cattle, horses or other ungulates can have significant benefits to the owner. This can be achieved by improving the fertility and / or fecundity of such non-human mammals. Currently, several methods or schemes have been proposed to improve reproductive performance based on the use of estradiol and other hormones such as GnRH (gonadotropin-releasing hormone), LH (luteinizing hormone), FSH (follicle stimulating hormone) or progestins (such as progesterone) to simulate natural or near-natural levels of hormones present in the target species. Some of these schemes include steps to synchronize ovulation in females to promote stimulation, growth and ovulation of follicles in a synchronized manner, thereby allowing artificial conception at a fixed time, avoiding the necessity of estrus detection. These so-called synchronization schemes are widely used in commercial dairy and beef herds around the world.

[0003] A kind of treatment method in the most common treatment method for making follicular wave appear synchronously involves the use of different types of estrogen. However, this steroid hormone cannot be used in many parts of the world, and alternative methods have been developed to control the follicular phase and the luteal phase, with the purpose of improving the superovulation process of embryo donor cows in countries where estradiol cannot be obtained. A method that seems to be a promising option is to initiate FSH treatment when new follicular wave appears after ovulation induced by GnRH. In addition, because the half-life of currently available FSH products is usually shorter (about 6 hours), the superovulation scheme involves FSH treatment twice a day in 4 to 5 days, which is quite time-consuming, laborious and prone to error. Therefore, the standard superovulation method is based on multiple injections of FSH when follicular wave appears, and follicular wave appears and usually occurs in 8 to 12 days after the estrus of natural occurrence. This method is called "based on estrus", and although it is effective in terms of embryo generation, it is restricted by the stage of the estrus cycle, and estrus detection is needed. Subsequent studies have found that fertilization rates are significantly lower in "estrus-based" protocols compared to modern synchronized protocols that allow for a more optimal interval from conception to ovulation.

[0004] Studies have been conducted to determine if extending the FSH stimulation regimen can increase the ovulatory response. The authors appear to have concluded that extending the FSH injection cycle from 4 consecutive days to 7 consecutive days may maintain follicle growth and allow more follicles to acquire the ability to ovulate. More studies are being conducted to confirm this hypothesis in different cattle breeds.

[0005] In another reported protocol, cows were treated with FSH over a 3-day period instead of a 4-day period, and the last 2 injections on day 4 were replaced by 2 injections of eCG (equine chorionic gonadotropin) (Barros et al., 2008). This treatment appears to increase the number of embryos, although the treatment time is still long. In addition, eCG is a fairly long molecule and has been described to induce antibody formation when used repeatedly, and may limit the widespread / frequent use of these types of protocols.

[0006] In an attempt to reduce the number of FSH injections, an alternative has been developed in which FSH is embedded in a slow-release polymer (hyaluronic acid). Using this slow-release formulation, a single injection is given after an "estrus-based" superovulation protocol. However, the authors concluded that hyaluronic acid is too viscous and a single intramuscular injection results in a low superovulation response (Bo et al., 2010).

[0007] EP2134165 proposes a method for increasing reproduction using single-chain FSH in which two subunits are covalently linked via a peptide linker, which is administered in a single dose of 5 μg-150 μg.

[0008] WO2014 / 076231 reports the use of a single dose of 50 μg or 100 μg of an RbFSH analogue to improve reproductive performance. Summary of the invention

[0009] In one aspect, the present invention relates to methods and compositions and uses for inducing, causing or stimulating superovulation ("SO") in bovine cows using a one-time administration of follicle stimulating hormone ("FSH"). The present invention also relates to such methods and compositions and uses for improving conception rates in bovine cows.

[0010] In another aspect, the present invention relates to methods and compositions and uses for increasing or improving egg collection ("OPU") in bovine cows using a one-time administration of follicle stimulating hormone ("FSH"). The present invention also relates to such methods and compositions and uses in artificial insemination or in vitro fertilization methods of bovine cows.

[0011] By conducting various tests and studies, the inventors have found that in cattle cows, FSH using a specific dosage range and schedule can improve ovulation or oocyte production. Specifically, by testing the efficacy of FSH according to various schemes and in different types of cows, it has been found that the response varies depending on the type of animal and the expected type of effect. More specifically, in cattle cows, contrary to previous reports in other types of cows, it was surprisingly found that a single administration dose of between 200 μg and 400 μg, most preferably between 250 μg and 350 μg of FSH can obtain the most effective superovulation. In view of the previous report that the efficacy is best when the dosage is about 50 μg, such a discovery is surprising. In addition, the inventors were also surprised to find that the FSH dosage is about 100 μg to 200 μg to obtain the best efficacy of allowing or improving the collection of cattle cow eggs, so the dosage is different from the dosage suitable for SO. Therefore, surprisingly, there is variability between the subtypes of cows, and there is variability in the best performance of cows of the same subtype in SO or OPU. Therefore, no conclusions can be drawn from the prior art to identify the most effective conditions for inducing SO or OPU in cattle.

[0012] In one aspect, the present invention arises from the discovery that high superovulation can be induced in bovine cows by a single administration of FSH at a dose of between 200 μg and 400 μg, particularly between 300 μg and 400 μg. This discovery is surprising in view of earlier reports of best efficacy with much lower doses (e.g., 50 μg), and in view of further data obtained by the inventors showing that the most effective OPU process is achieved in bovine cows with a single dose of about 100 μg.

[0013] In another aspect, the present invention arises from the discovery that high OPU can be induced or achieved in bovine cows by a single administration of FSH at a dose of between 100 μg and 200 μg. This discovery is surprising in view of earlier reports of OPU without injection of FSH, and in view of further data obtained by the present inventors showing that the most effective SO is induced in bovine cows with a single dose of about 200 μg-400 μg.

[0014] A particular object of the invention relates to the use of FSH or a composition comprising FSH for inducing, causing or stimulating superovulation in bovine cows, wherein said FSH is administered to said bovine cow once during the estrus cycle in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0015] Another object of the invention is a method for inducing or stimulating or increasing superovulation in bovine cows, comprising administering FSH to said bovine cow once during the estrus cycle in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0016] Another object of the invention is FSH for use in a method for inducing or stimulating or increasing superovulation in bovine cows, said method comprising administering said FSH once during the estrous cycle to said bovine cow in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0017] Another specific object of the invention relates to the use of FSH or a composition comprising FSH in a method for oocyte collection in bovine cows, wherein said FSH is administered to said bovine cows at a dose of between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0018] Another preferred object of the present invention relates to the use of FSH or a composition comprising FSH in a method for oocyte collection in bovine cows, wherein said FSH is administered once during the estrus cycle to said bovine cow at a dose of between 100 μg and 200 μg. Preferably, the method further comprises a subsequent step of collecting oocytes.

[0019] Another object of the invention is a method for oocyte collection in bovine cows, comprising administering FSH to said bovine cows at a dose between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0020] Another preferred object of the present invention is a method for oocyte collection in bovine cows, comprising administering FSH to said bovine cow once during the estrus cycle at a dose between 100 μg and 200 μg. Preferably, the method further comprises a subsequent step of collecting oocytes.

[0021] Another object of the invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH to said bovine cow at a dose of between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0022] Another preferred object of the present invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH once during the estrus cycle to said bovine cow at a dose between 100 μg and 200 μg. Preferably, the method further comprises the subsequent step of collecting oocytes.

[0023] Preferably, the FSH is recombinant FSH (rFSH).

[0024] As will be further disclosed, in a preferred embodiment, the FSH comprises a bovine FSH alpha subunit and a bovine FSH beta subunit covalently linked to each other via a peptide linker.

[0025] The present invention can be used with any bovine cow, particularly any mature bovine cow, such as a young adult animal or an elderly animal, a dairy cow or a beef cow. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : The nucleotide and amino acid sequence of FSH used in the present invention. The CTP linker sequence connecting the β subunit and the α subunit is underlined. The underlined and bold nucleotides encode the signal peptide.

[0027] Figure 2 : Graph showing the number of small (<6 mm), medium (6 mm-10 mm) and large (>10 mm) follicles in heifer donors (Holstein cows) subjected to OPU for superstimulation with different doses of rFSH.

[0028] Figure 3 : Graph showing the number of small (<6 mm), medium (6 mm-10 mm) and large (>10 mm) follicles from lactating Holstein cow donors subjected to OPU for superstimulation with different doses of rFSH. DETAILED DESCRIPTION

[0029] The present invention provides methods for inducing or improving SO or OPU in domestic cows. The present invention uses FSH in a single injection per cycle schedule under conditions that can most effectively induce or increase superovulation, high-quality embryo production, high-quality oocyte production, pregnancy rate and / or OPU.

[0030] definition

[0031] The term "superovulation" is generally characterized by a high / increased number of ovulating follicles and / or fertile eggs compared to conventional ovulation. Typically, in the context of the present invention, the term superovulation is characterized by the induction of at least 5 (preferably at least 6) embryos (grade 1+grade 2+grade 3) that can be frozen per bovine cow, wherein the percentage of embryo viability is higher than 45%, preferably higher than 50%, and at least 80% are ovulating cows. The most preferred superovulation according to the present invention is characterized by at least 6 embryos (grade 1+grade 2+grade 3) that can be frozen per bovine cow, wherein the percentage of embryo viability is higher than 50%, and at least 80% are ovulating cows, more preferably at least 100% are ovulating cows.

[0032] The term "egg collection" refers to a protocol comprising the steps of collecting oocytes from a cow, usually for the purpose of artificial insemination or conception. In order to achieve OPU (and appropriate subsequent fertilization / conception), it is desirable to collect high-quality oocytes that are most likely to be fertilized into high-quality embryos. In the context of the present invention, the improved OPU is preferably characterized by an oocyte recovery rate of at least 60%, preferably at least 65%, and a large follicle level of at least 3%, preferably at least 4%, at least 5% or at least 6%. Preferably, an oocyte recovery rate of at least 60%, preferably at least 65%, is obtained, and (i) less than 50%, more preferably less than 40%, or even less than 35% of the follicles are small follicles, and / or (ii) greater than 40%, preferably greater than 50% of the follicles are medium follicles, and / or (iii) greater than 5%, preferably greater than 6% of the follicles are large follicles. The most optimal OPU is achieved with the following percentages of small to medium to large follicles: large: >6%; medium: >50%; small: <35%.

[0033] In the context of the present invention, the term "fertility" or "fertile" refers to the ability to produce oocytes that are capable of fertilization.

[0034] In the context of the present invention, the term "fertile" or "fertility" refers to the ability to complete a pregnancy.

[0035] The term "pregnant" refers to a non-human mammal or some of a population of non-human mammals that is currently in the pregnancy stage or has been conceived and may be in the pregnancy stage.

[0036] As used herein, the term "estrus" refers to the period during which a non-human mammal is most likely to become pregnant. Estrus can be detected or monitored by the behavioral displays of a non-human mammal when in estrus, including exhibiting persistent estrus.

[0037] "Implantation" refers to the introduction of semen by any method known in the art, including but not limited to natural and artificial insemination (AI) and in vitro fertilization (IVF).

[0038] An animal "population" is any group of at least 2 non-human mammals, such as a herd of cattle or sheep. A typical population contains between 2 and 5000 animals.

[0039] The term "administration" refers to all routes of administration, such as oral administration, intestinal mucosal administration, parenteral administration or transdermal administration. Preferably, the administration route is injection.

[0040] In the context of the present invention, the term "follicular synchronization" refers to the synchronization of the occurrence of follicular waves.

[0041] The term "Bos" refers to a subtype of cattle of the cow. This subtype is also known as common cattle. Bos is a different subtype of cow than the zebu (Bosindicus), also known as the domestic zebu.

[0042] For example, examples of cattle breeds include, for example, Red Angus, Black Angus, Holstein, Friesian, Charolais, Limousin, Wagyu, Simmental, Jersey, Brown Swiss, Hereford / Angus, crossbreds, and mongrel cattle.

[0043] For instance, examples of zebu cattle include, for example, Nellore cattle, Afrika cattle, Bachaur cattle, Bolan cattle, Deoni cattle, Wind cattle, Gaolao cattle, Gir cattle, Guzera cattle, Indo-Brazilian cattle, Malvi cattle, Nagori cattle, Nimari cattle, crossbred cattle and hybrid cattle.

[0044] The term "de-milking cow" refers to a dairy cow that is at the stage in its lactation cycle where it stops producing milk prior to calving.

[0045] FSH

[0046] Follicle stimulating hormone belongs to the fertility hormone class. FSH is composed of two chains (or subunits) called α and β. Under physiological conditions, the two subunits are connected by non-covalent interactions. The nucleotide and amino acid sequences of FSH from various species have been disclosed and can be obtained in public databases, such as bovine FSH (Kim KE et al., 1988, "Nucleotide sequence of the bovine gene for follicle-stimulating hormone beta-subunit", DNA 1988, 7 (4): 227-233) or human FSH: Gene Bank Number: CAA43996.1.

[0047] The present invention preferably utilizes recombinant FSH (rFSH), eg, FSH produced by and / or engineered using recombinant techniques.

[0048] In a specific embodiment, FSH is recombinant bovine FSH or its variant. Variant refers to FSH with 1 to 10, preferably 1 or 2 or 3 amino acid modifications (e.g., substitutions, additions, deletions) compared to the reference sequence, which modifications do not substantially change the FSH activity. Such modifications can be carried out, for example, to improve the half-life of FSH.

[0049] In a preferred embodiment, the FSH used in the present invention is FSH having an α subunit and a β subunit covalently linked together. This preferred FSH may also be designated as "single-chain" FSH.

[0050] In a more preferred embodiment, the present invention uses the recombinant FSH that α subunit is covalently connected to β subunit by peptide linker.Preferably, α subunit is the α subunit of bovine FSH (or its variant), and / or β subunit is the β subunit of bovine FSH (or its variant).This peptide linker can be any peptide linker that does not substantially affect the conformation or activity of FSH.In a preferred embodiment, this linker is a CTP linker, for example, comprising a linker of the sequence of the carboxyl terminal peptide of human chorionic gonadotropin as described in, for example, US6,242,580 and / or US2008 / 0312151.In another preferred embodiment, this linker is a peptide comprising a (G4S) sequence, which can be repeated once or several times.

[0051] A specific example of the alpha subunit of bovine FSH is represented by SEQ ID NO: 1. The sequence and variants thereof, such as variants having 1 or 2 or 3 amino acid modifications (eg, substitutions, additions, deletions) that do not substantially alter the activity of FSH, are suitable for use in the present invention.

[0052] A specific example of the beta subunit of bovine FSH is represented by SEQ ID NO: 2. The sequence and variants thereof, such as variants having 1 or 2 or 3 amino acid modifications (eg, substitutions, additions, deletions) that do not substantially alter the activity of FSH, are suitable for use in the present invention.

[0053] In a preferred embodiment, the FSH used in the present invention has a half-life in physiological solution of at least 15 hours, preferably at least 20 hours.

[0054] Figure 1The cDNA and amino acid sequences of specific recombinant FSH used in the present invention are provided in SEQ ID NO:3 and SEQ ID NO:4. The first portion (amino acids 1-129) of SEQ ID NO:3 corresponds to the sequence of the bovine FSH β subunit (SEQ ID NO:2), the middle portion corresponds to the carboxyl terminal peptide linker (amino acids 130-157), and the third portion (amino acids 158-253) corresponds to the sequence of the bovine FSH α subunit (SEQ ID NO:1).

[0055] In a specific embodiment, the present invention uses a recombinant FSH having, comprising, essentially consisting of or consisting of: (i) the amino acid sequence of SEQ ID NO: 3, or (ii) an amino acid sequence having at least 90%, preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 3 over its entire length.

[0056] FSH is preferably isolated, and more preferably substantially pure, for example, having a purity level of at least 95%, more preferably at least 97%, 98% or 99%.

[0057] In a specific embodiment, FSH or recombinant FSH is used in a composition comprising a suitable pharmaceutical preparation. The pharmaceutical preparation may include one or more excipients or carriers, such as excipients suitable for liquid preparations or solid preparations. The preparation is typically a liquid preparation, such as a solution or suspension, preferably suitable for injection. The liquid preparation may contain any suitable diluent, such as a saline solution, a buffer solution, a physiological solution, etc. The example of such a suitable diluent includes phosphate buffered saline. Additives may be present in the preparation, such as preservatives, stabilizers, etc. In a specific embodiment, the preparation is a liquid preparation with a dosage of about 50 μg FSH / ml.

[0058] Superovulation

[0059] The present invention relates to a method for inducing / increasing / stimulating superovulation and / or improving pregnancy rates in bovine cows using FSH.

[0060] As discussed above, in one aspect, the present invention arises from the discovery that high superovulation can be induced in bovine cows by a single administration of FSH at a dose of between 200 μg and 400 μg. This discovery is surprising in view of earlier reports of best efficacy using much lower doses (e.g., 50 μg) in other types of cows, and in view of further data contained in the present application showing that the most effective OPU process in bovine cows is achieved using a single dose of about 100 μg.

[0061] Therefore, a particular object of the present invention relates to the use of FSH or a composition comprising FSH for inducing, causing or stimulating superovulation in bovine cows, wherein said FSH is administered to said bovine cow once during the estrus cycle in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0062] Another object of the invention is a method for inducing or stimulating or increasing superovulation in bovine cows, comprising administering FSH to said bovine cow once during the estrus cycle in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0063] Another object of the invention is FSH for use in a method for inducing or stimulating or increasing superovulation in bovine cows, said method comprising administering said FSH once during the estrous cycle to said bovine cow in a dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg.

[0064] In a preferred embodiment, which may be combined with any other specific and / or preferred embodiment, FSH is administered at a dose of 200 μg, 220 μg, 230 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 370 μg or 400 μg, preferably at a dose of between 250 and 350 μg, even more preferably at a dose of 260, 270, 280, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg or 340 μg, or at a dose of between 300 and 400 μg, such as at a dose of 350, 360, 370, 380, 390 μg.

[0065] The data presented in this application show that this dosage range results in greater than 80% of ewes ovulating, often 100% of ewes ovulating, and can induce a large number of viable embryos that can be frozen, resulting in improved conception / pregnancy rates.

[0066] In a specific embodiment that can be combined with any other specific and / or preferred embodiment, FSH is administered once between the 7th and 12th day of the estrous cycle, preferably between the 8th and 11th day, such as on the 8th, 9th, 10th or 11th day of the estrous cycle, even more preferably on the 9th or 10th day. Those skilled in the art can easily detect and / or monitor the estrous cycle. In some cases, if appropriate, ultrasound technology can be used to determine the start of the estrous cycle according to methods known in the art (see also the experimental part).

[0067] In a particular embodiment, the invention relates to a method or use comprising administering FSH to a bovine cow, preferably once by injection, more preferably by intramuscular injection, at a dose of between about 200 μg and about 400 μg between days 7 and 12 of the estrus cycle of said cow.

[0068] In another specific embodiment, which may be combined with any other specific and / or preferred embodiment, FSH is administered to synchronized cows (e.g., cows that have been previously treated to synchronize follicles). Methods of synchronizing cows are known in the art per se, as discussed in further sections below.

[0069] FSH or a composition comprising FSH may be administered by any suitable route and in any suitable manner. Preferably, FSH is administered by systemic administration, particularly injection, such as intramuscular injection, intravenous injection or subcutaneous injection. The preferred route of administration is intramuscular injection.

[0070] In a preferred embodiment, the composition or method or regimen of the present invention uses a single dose of between 250 μg and 350 μg of FSH, preferably in the form of a liquid formulation suitable for injection, preferably intramuscular injection.

[0071] In another preferred embodiment, the composition or method or regimen of the present invention uses a single dose of about 300 μg of FSH, preferably in the form of a liquid formulation suitable for injection, preferably intramuscular injection.

[0072] The results presented in the experimental section indicate that this single administration leads to the production of a large number of fertile embryos capable of implantation.

[0073] In another specific embodiment, which may be combined with any other specific and / or preferred embodiment, the method, use or regimen of the invention further comprises the step of impregnating the cow.

[0074] In this respect, an object of the invention is a method for impregnating a bovine cow, comprising (i) inducing or stimulating superovulation according to the invention and (ii) impregnating the cow.

[0075] In a specific embodiment, the present invention relates to a method or use comprising:

[0076] (a) administering FSH to a bovine cow, preferably once by injection, more preferably by intramuscular injection, between day 7 and day 12 of the estrus cycle of said cow at a dose of between about 200 μg and about 400 μg, in particular between 300 μg and 400 μg; and

[0077] (b) The cow is preferably impregnated by artificial insemination.

[0078] Preferably, conception is performed or allowed to occur close to ovulation. In a preferred embodiment, conception is performed (or allowed to occur) between 3 and 8 days, more preferably between 4 and 7 days after the injection of FSH in step (a). Preferably, conception is performed by artificial insemination.

[0079] Can optionally use one or more additional hormones, such as luteinizing hormone, chorionic gonadotropin and / or prostaglandin, to further improve pregnancy.In one embodiment, except using FSH, also cow is used prostaglandin.Can single dose be used prostaglandin by injection usually, or use this prostaglandin with multiple dose interval several hours.In one embodiment, use a dosage, dosage is between 5mg and 50mg.Preferably use prostaglandin in the 2nd day, 3 days or 4 days after FSH uses.

[0080] In another embodiment, in addition to the administration of FSH (and optional prostaglandins), GnRH is also administered to the cow. GnRH is administered in a single dose, or generally in two consecutive doses, which can be administered several hours (or 1 day) apart. In one embodiment, the method / use comprises administering GnRH to the cow preferably on the 4th, 5th or 6th day after FSH administration, and even more preferably on the 5th and 6th day after FSH administration. The total dose of GnRH is preferably between 50 μg and 200 μg. If two consecutive administrations are performed, preferably 100 μg is administered each time. GnRH is preferably administered by injection.

[0081] In a specific embodiment which may be combined with any other specific and / or preferred embodiment, the method, use or regimen of the invention therefore further comprises administering a prostaglandin to said cow, preferably on day 2, 3 or 4 after the administration of FSH.

[0082] In another specific embodiment that may be combined with any other specific and / or preferred embodiment, the method, use or regimen of the present invention further comprises administering GnRH to the cow, preferably on day 4, day 5 or day 6 after FSH administration, even more preferably on day 5 and day 6 after FSH administration.

[0083] In this regard, in a specific embodiment, the present invention relates to a method or use or regimen as described above, comprising:

[0084] (a) administering FSH to a bovine cow, preferably once by injection, more preferably by intramuscular injection, between day 7 and day 12 of the estrus cycle of said cow in a dose of between about 200 μg and about 400 μg, in particular between 300 μg and 400 μg;

[0085] (b) administering prostaglandins and / or GnRH to the cow; and

[0086] (c) The cow is preferably impregnated by artificial insemination.

[0087] As discussed above, the FSH used in the present invention preferably comprises a bovine FSH α subunit and a bovine FSH β subunit covalently linked to each other via a peptide linker. In a specific embodiment, the present invention uses FSH having, comprising, consisting essentially of, or consisting of (i) the amino acid sequence of SEQ ID NO: 3, or (ii) an amino acid sequence having at least 90%, preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 3 over its entire length, and retaining the FSH activity or characteristics as defined above.

[0088] In another specific embodiment, the method / use / scheme comprises:

[0089] (a) injecting a bovine cow with FSH comprising a bovine FSH α subunit and a bovine FSH β subunit covalently linked to each other via a peptide linker, wherein the FSH is administered once between day 8 and day 11 of the estrus cycle of the cow in a dose of between about 200 μg and about 400 μg, in particular between 300 μg and 400 μg, more preferably by intramuscular injection;

[0090] (b) optionally administering prostaglandins and / or GnRH to the cow; and

[0091] (c) impregnating the cow, preferably by artificial insemination, 3 to 8 days, more preferably 4 to 7 days, after the injection of FSH in step (a).

[0092] In a specific embodiment, the method / use / regimen is performed on a synchronized herd of cows (eg, a herd of cows that have been treated to synchronize their follicles).Such an embodiment may obviate the need to detect or monitor estrus.

[0093] The use / method / regimen of the invention as defined above may be implemented during one, several or every estrus cycle of a bovine cow.

[0094] Egg collection

[0095] In another aspect, the present invention arises from the discovery that high OPU can be induced or achieved in bovine cows by a single administration of FSH at a dose between 100 μg and 200 μg. This discovery is surprising in view of earlier reports of OPU without injection of FSH, and in view of further data presented in the present application, which further data show that the most effective SO is induced in bovine cows with a single dose of about 200 μg-400 μg. Specifically, the inventors have shown that high OPU can be induced or achieved in bovine heifers by a single administration of FSH at a dose of 100 μg. The inventors further show that high OPU can be induced or achieved in bovine lactating cows by a single administration of FSH at a dose of 150 μg.

[0096] Therefore, a specific object of the invention relates to the use of FSH or a composition comprising FSH in a method for oocyte collection in bovine cows, wherein said FSH is administered to said bovine cow at a dose of between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0097] The invention also relates to the use of follicle stimulating hormone (FSH) in the preparation of a treatment for oocyte collection application in bovine cows, wherein said FSH is administered to said bovine cow in a dose of between 100 μg and 200 μg, between 100 μg and 150 μg, in particular 100 μg or 150 μg.

[0098] Therefore, a preferred object of the present invention relates to the use of FSH or a composition comprising FSH in a method for oocyte collection in bovine cows, wherein said FSH is administered once during the estrus cycle to said bovine cow in a dose of between 100 μg and 200 μg. The method also comprises the subsequent step of collecting oocytes.

[0099] Another preferred object of the present invention relates to the use of FSH or a composition comprising FSH in a method for oocyte collection in bovine cows, wherein said FSH is administered once during the estrus cycle to said bovine cow at a dose of 100 μg or 150 μg. The method also comprises a subsequent step of collecting oocytes.

[0100] Another object of the invention is a method for oocyte collection in bovine cows, comprising administering FSH to said bovine cows at a dose between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0101] Another object of the present invention is a method for oocyte collection in bovine cows, comprising administering FSH to said bovine cows at a dose of 100 μg or 150 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0102] Another preferred object of the present invention is a method for oocyte collection in bovine cows, comprising administering FSH to said bovine cow once during the estrus cycle at a dose between 100 μg and 200 μg. The method also comprises a subsequent step of collecting oocytes.

[0103] Another preferred object of the present invention is a method for oocyte collection in bovine cows, comprising administering FSH to the bovine cow once during the estrus cycle at a dose of 100 μg or 150 μg. The method also comprises a subsequent step of collecting oocytes.

[0104] Another object of the invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH to said bovine cow at a dose of between 100 μg and 200 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0105] Another object of the invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH to said bovine cow at a dose of 100 μg or 150 μg. Preferably, FSH is administered only once, followed by oocyte collection.

[0106] Another preferred object of the present invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH once during the estrus cycle to said bovine cow at a dose between 100 μg and 200 μg. The method also comprises the subsequent step of collecting oocytes.

[0107] Another preferred object of the present invention is FSH for use in a method for oocyte collection in bovine cows, said method comprising administering said FSH once during the estrus cycle at a dose of 100 μg or 150 μg to said bovine cow. The method also comprises the subsequent step of collecting oocytes.

[0108] In a preferred embodiment that can be combined with any other specific and / or preferred embodiment, with a dosage of 100 μ g, 110 μ g, 120 μ g, 130 μ g, 140 μ g, 150 μ g, 160 μ g, 170 μ g, 180 μ g, 190 μ g or 200 μ g, preferably with a dosage between 100 μ g and 150 μ g, even more preferably with a dosage of about 100 μ g. In a specific embodiment, FSH is administered at a dosage of 100 μ g. In another specific embodiment, FSH is administered at a dosage of 150 μ g.

[0109] The data presented in this application show that this dose range induces a high percentage of large follicles with a high oocyte recovery rate, resulting in improved fertilization.

[0110] Through specific work, the inventors have found that administration of a single dose of 100 μg is particularly effective for OPU in bovine heifers. Similarly, the inventors have found that administration of a single dose of 150 μg is particularly effective for OPU in lactating bovine cows.

[0111] Those skilled in the art can easily detect and / or monitor the estrous cycle.In some cases, if appropriate, ultrasound technology can be used to determine the start of the estrous cycle according to methods well known in the art (see also the experimental part).

[0112] In a particular embodiment, the invention relates to a method or use for OPU comprising administering FSH to a bovine cow, preferably once by injection, more preferably by intramuscular injection, at a dose of between about 100 μg and about 200 μg.

[0113] In a preferred embodiment, the present invention relates to a method or use for OPU comprising administering FSH to a bovine heifer, said FSH being administered once at a dose of 100 μg, preferably by injection, more preferably by intramuscular injection.

[0114] In another preferred embodiment, the present invention relates to a method or use for OPU, comprising administering FSH to a lactating bovine cow, said FSH being administered once at a dose of 150 μg, preferably by injection, more preferably by intramuscular injection.

[0115] FSH or a composition comprising FSH may be administered by any suitable route and in any suitable manner. Preferably, FSH is administered by systemic administration, particularly injection, such as intramuscular injection, intravenous injection or subcutaneous injection. The preferred route of administration is intramuscular injection.

[0116] In a preferred embodiment, the composition or method or regimen of the present invention uses a single dose of between 100 μg and 200 μg of FSH, preferably in the form of a liquid formulation suitable for injection, preferably intramuscular injection.

[0117] In another preferred embodiment, the composition or method or regimen of the present invention uses a single dose of 100 μg of FSH, preferably in the form of a liquid formulation suitable for injection, preferably intramuscular injection.

[0118] In another preferred embodiment, the composition or method or regimen of the present invention uses a single dose of 150 μg of FSH, preferably in the form of a liquid formulation suitable for injection, preferably intramuscular injection.

[0119] The results presented in the experimental section indicate that this single administration leads to the production of large numbers of highly fertile oocytes.

[0120] In another specific embodiment, which may be combined with any other specific and / or preferred embodiments, the OPU method, use or regimen of the invention further comprises the step of collecting oocytes.

[0121] In this respect, an object of the present invention is a method for obtaining oocytes from a bovine cow, comprising (i) administering to said bovine cow once FSH at a dose of between 100 μg and 200 μg, preferably by injection, more preferably by intramuscular injection; and (ii) collecting oocytes.

[0122] In a specific embodiment, the bovine cow is a heifer and the dose of FSH is 100 μg.

[0123] In another specific embodiment, the bovine cow is a lactating cow and the dose of FSH is 150 μg.

[0124] Preferably, oocyte collection is performed on the 2nd, 3rd, 4th or 5th day after FSH administration. The data obtained by the inventors show that a high recovery rate (greater than 60%, more preferably greater than 65%) and high-quality oocytes (large follicles account for more than 3%, preferably more than 4%, more than 5%, and even more preferably more than 6%) of the total follicles are obtained using the method of the present invention. This performance allows the collected oocytes to be successfully fertilized.

[0125] In another specific embodiment, which may be combined with any other specific and / or preferred embodiment, the OPU method, use or regimen of the invention further comprises a step of conception of the collected oocytes, typically in vitro conception (FIV).

[0126] In this respect, an object of the present invention is a method comprising (i) administering FSH once to a bovine cow at a dose of between 100 μg and 200 μg, preferably by injection, more preferably by intramuscular injection; (ii) collecting oocytes from the cow, preferably on the 2nd, 3rd, 4th or 5th day after the FSH administration; and (iii) fertilizing these oocytes, preferably in vitro.

[0127] In particular, an object of the present invention is a method comprising (i) administering FSH once to a bovine heifer at a dose of 100 μg, preferably by injection, more preferably by intramuscular injection; (ii) collecting oocytes from the cow, preferably on the 2nd, 3rd, 4th or 5th day after the FSH administration; and (iii) fertilizing these oocytes, preferably in vitro.

[0128] Another specific object of the present invention is a method comprising (i) administering FSH once to a lactating bovine cow at a dose of 150 μg, preferably by injection, more preferably by intramuscular injection; (ii) collecting oocytes from the cow, preferably on the 2nd, 3rd, 4th or 5th day after the FSH administration; and (iii) preferably fertilizing these oocytes in vitro.

[0129] In vitro conception can be performed at any time after collection. However, it is preferred that conception be performed between 6 hours and 3 days after collection. Alternatively, the oocytes can be frozen and then thawed for conception. The fertilized oocytes can then be cultured in vitro (until embryonic development) or impregnated according to methods known in the art.

[0130] As discussed above, the FSH used in the present invention preferably comprises a bovine FSH α subunit and a bovine FSH β subunit covalently linked to each other via a peptide linker. In a specific embodiment, the above method, use or regimen of the present invention uses FSH having, comprising, consisting essentially of or consisting of (i) the amino acid sequence of SEQ ID NO: 3, or (ii) an amino acid sequence having at least 90%, preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 3 over its entire length, and retaining the FSH activity or characteristics as defined above.

[0131] In another specific embodiment, the method / use / scheme comprises:

[0132] (a) injecting a bovine cow with FSH, said FSH comprising a bovine FSH alpha subunit and a bovine FSH beta subunit covalently linked to each other via a peptide linker, said FSH being administered once to said cow at a dose of between about 100 μg and about 200 μg, more preferably by intramuscular injection; and

[0133] (b) collecting oocytes from said cow, preferably between 2 days and 5 days after the administration of FSH.

[0134] In one specific embodiment, the bovine cow is a heifer and the FSH dose is 100 μg. In another specific embodiment, the bovine cow is a lactating cow and the FSH dose is 150 μg.

[0135] In a specific embodiment, the method comprises the further step (c) of fertilizing the collected oocytes in vitro.

[0136] Follicle synchronization

[0137] In a specific embodiment, the method / use / protocol of the present invention is performed on synchronized cows. In combination with the FSH treatment of the present invention, synchronization of the emergence of new follicle waves improves embryo production. In addition, by synchronizing the follicles, the need to monitor the estrus cycle is avoided.

[0138] Follicle growth is not continuous in cows, but appears in waves (2 to 4 waves per cycle). Each wave starts approximately when the dominant follicle in the previous wave obtains the maximum size, when many small follicles begin a period of rapid growth. From this group of follicles, one follicle is allowed to grow to a much larger size than the other follicles. This large follicle is called the dominant follicle because it has the ability to regulate and limit the growth of smaller follicles called inferior follicles. A few days after reaching the maximum size, the dominant follicle begins to degenerate and die. When the dominant follicle degenerates, its ability to limit other follicles decreases; therefore, a new wave of follicles is initiated. The consequence of this dynamic process is that follicles of all sizes, including at least one large follicle, are present on every day of the estrus cycle. Follicle wave synchronization gives the opportunity to treat all cows within a limited time, and thus obtains the economic benefits of pregnancy. After the synchronization of the estrus cycle, a high percentage of treated females show fertile, tightly synchronized estrus and ovulation.

[0139] In a specific embodiment, follicular synchronization is obtained by hormonal treatment, preferably with a gestagen, a gestagen-prostaglandin combination, a prostaglandin alone, a gestagen-estrogen combination, or a gonadotropin-prostaglandin combination with or without a gestagen.

[0140] In a specific embodiment, follicle synchronization is achieved by one of the following treatments:

[0141] -PGF2α or its analogs;

[0142] -GnRH+PGF2α

[0143] - a gestagen (such as progesterone), optionally in combination with estrogen or PGF2α or GnRH.

[0144] Specific dosages and / or regimens are disclosed in the art, for example, see Thatcher et al., 2001 (American Association of Bovine Practitioner, AABP), Vancouver, 95-105); Diskin et al., 2001 (occasional publication n°26, pl75, British society of Animal Science); or Pursley et al., 1995 (Theriogenology 44 p915). In addition, progestins may be administered using specific devices such as implants (e.g., PRID by Ceva Sante Animale).

[0145] In another embodiment, follicular synchronization can be achieved by follicular ablation. Ablation of follicles refers to the elimination, removal or destruction of at least one follicle. Follicular ablation refers to physical methods of follicle ablation, such as cauterization or ultrasound-guided transvaginal follicular aspiration at random stages of the estrous cycle (Bergfelt, 1997). Ablation can be directed to all follicles or only one or two of the largest follicles (Baracaldo et al., 2000) to ensure that at least the dominant follicle is suppressed.

[0146] In a particular embodiment, the methods, uses, regimens of the invention may therefore comprise a prior step of synchronizing follicles in a population of bovine cows, such as, in particular, by a method comprising the steps of treating bovine cows (or a population of bovine cows) with a progestin, a progestin-prostaglandin combination, a prostaglandin, a progestin-estrogen combination or a gonadotropin-prostaglandin combination with or without a progestin.

[0147] In a specific embodiment, the present invention therefore relates to a method, use, or regimen comprising:

[0148] (a) treating a bovine cow (or a group of bovine cows) with a gestagen, a gestagen-prostaglandin combination, a prostaglandin, a gestagen-oestrogen combination, or a gonadotropin-prostaglandin combination with or without a gestagen, to synchronize ovarian follicles; or providing a bovine cow (or a group of bovine cows) that has been treated with a gestagen, a gestagen-prostaglandin combination, a prostaglandin, a gestagen-oestrogen combination, or a gonadotropin-prostaglandin combination with or without a gestagen, to synchronize ovarian follicles;

[0149] (b) administering to the synchronized bovine cows (or herd of bovine cows) a single dose of between 200 μg and 400 μg, in particular between 300 μg and 400 μg, of FSH; and

[0150] (c) preferably impregnates the cow close to ovulation, more preferably 2 to 7 days, even more preferably 4 to 6 days after administering step (b).

[0151] Further aspects and advantages of the invention will be disclosed in the following experimental section, which illustrates the claimed invention.

[0152] Example

[0153] Example 1: One dose of bovine follicle stimulating hormone induces superovulation in mature beef cattle of the domestic bovine subtype

[0154] The aim of this study was to evaluate the efficacy of bovine follicle-stimulating hormone on superovulation in mature beef cattle of the domestic subtype and to design an optimal protocol.

[0155] The study was conducted in full compliance with GCP standards, VICH GL9 "Guide for Industry: Good Clinical Practice" and CVM GFI 56 "Guide for Industry: Development of Clinical Effectiveness and Target Animal Safety Test Plans". The study was a single-blind parallel study design with five treatment groups. Fifty mature female beef cows were randomly assigned to five groups treated with recombinant single-chain bovine FSH (50μg, 100μg, 200μg, 300μg and 400μg; 10 cows per group) and compared with historical controls.

[0156] After treatment on day 0 (corresponding to day 9 / 10 of the estrous cycle), cows were given prostaglandins on day 3 to lyse the corpora lutea, GnRH on days 5 and 6 to induce ovulation, and artificial insemination was performed on day 6. Embryos were harvested and evaluated on day 12. Parameters measured included the number and size of follicles and the number of corpora lutea on day 3, the number of corpora lutea on day 12, and the number and quality of embryos on day 12.

[0157] IVP

[0158] The FSH used was a recombinant single-chain bovine FSH named RIPA follitropin alpha (bovine). RIPA follitropin alpha (bovine) is a glycoprotein whose main structure consists of the β subunit of bovine FSH covalently linked to the α subunit of bovine FSH. The amino acid sequence of RIPA follitropin alpha (bovine) is provided in the form of SEQ ID NO: 3.

[0159] FSH is formulated into a veterinary composition having the following ingredients:

[0160] Active ingredient: RIPA Follicle-stimulating Hormone Alpha (Bovine)

[0161] Amount: 476μg

[0162] Concentration: 4.46mg / mL

[0163] Excipient: Phosphate buffered saline

[0164] Dosage form: Injectable solution

[0165] Packaging: 10ml glass vial

[0166] Storage conditions: Store at 2°C-8°C until use

[0167] Treatment Groups and Randomization Procedure

[0168] On day 3, the health of the cows was assessed by history and physical examination (including heart rate, respiratory rate and weight). At the same time, manual inspection and ultrasound examination were performed to confirm the normal reproductive anatomy and the presence of corpus luteum of each cow. Fifty (50) reproductively mature domestic beef cows with normal corpus luteum and meeting all registration criteria were registered, ranging in age from 24 months to 84 months and ranging in weight from 1015 pounds (461 kg) to 1380 pounds (627 kg).

[0169] On Day 1, cows were randomly assigned to treatment groups using a computer-generated randomization table provided by the sponsor. Enrolled cows were treated with RIPA follitropin alfa (bovine) on Day 0 (i.e., Day 9 or 10 of the estrous cycle) with one of the following treatments shown in Table 1:

[0170] Table 1 Treatment groups

[0171]

[0172] IVP was administered once intramuscularly on day 0. The IVP was administered on the right side of the neck using a sterile plastic disposable 10 ml syringe fitted with a sterile disposable 1.5 inch 18 gauge needle.

[0173] All 50 cows received an IM injection of 25 mg of prostaglandin 72 hours (+ / - 2 hours) after RIPA follitropin alpha (bovine) treatment. Simultaneously with this injection of prostaglandin, an ultrasound examination was performed to evaluate the number and size of follicles (small = 2 mm-3 mm, medium = 4 mm-8 mm, and large ≥ 9 mm) and the number of corpora lutea on each ovary.

[0174] Forty-eight (48) hours (+ / - 2 hours) after prostaglandin treatment, each cow received an intramuscular injection of 100 μGnRH. Prostaglandin treatment and GnRH treatment were administered to the right and left hips, respectively.

[0175] About 32 hours after prostaglandin treatment, the estrus of all registered cows was observed. This started during the afternoon of the 4th day (4pm-6:21pm; Deviation 1). All cows were accepted by GnRH treatment on the 5th day morning (7am-10am). Two cows were in estrus on the 4th day afternoon and artificially conceived on the 5th day morning (7am-10am). Those identical cows were artificially conceived on the 5th day afternoon (4pm-6:07pm; Deviation 2). Forty-eight cows were only artificially conceived once on the 5th day afternoon, and received the second artificial conceive on the 6th day morning (7am-10am). Semen was derived from a single bull (Money Talks-SEKGenetics, Galesburg, KS 66740). On the 12th day of the study (7 days after the first artificial conception), manual inspection was performed to determine the CL count on each ovary.

[0176] Embryos were harvested (flushed). After embryo harvest on study day 12, cows were treated with prostaglandins to promote the restoration of cyclicity. Embryos from each cow were treated separately throughout the evaluation process. Embryos were graded according to the International Embryo Transfer Society's criteria (IETS Manual, 4th edition).

[0177] result

[0178] A total of 50 beef cattle (breeds including Red Angus, Black Angus, Hereford / Angus cross, and Crossbred) were enrolled in the study. The age and weight of the cows by dose group are summarized in Table 2.

[0179] Table 2: Summary of cow population statistics and ovulation results (mean and standard deviation)

[0180]

[0181] Prior to treatment with prostaglandins, ovaries were evaluated by ultrasound on day 3 to evaluate follicle growth in response to each dose of RIPA follitropin alpha (bovine). Table 3 shows the average number of follicles observed for each treatment group. There was a dose effect in response to RIPA follitropin alpha (bovine), as the average number of follicles tended to increase with increasing doses of RIPA follitropin alpha (bovine). The ovaries were also evaluated on day 12 to determine the number of corpora lutea produced, thereby estimating the ovulation rate after treatment with GnRH. Table 3 also lists the average number of corpora lutea for each treatment group. The ovulatory follicles in the lowest (50μg and 100μg) dose groups were less than those in the higher (200μg-400μg) dose groups. As part of the embryo evaluation process, the total number of eggs was counted, also on day 12. In the higher dose groups, the total number of eggs was higher.

[0182] Table 3: Follicular development and ovulation rate (mean and standard deviation)

[0183]

[0184] Embryos were collected and evaluated on day 12. Table 4 below lists the means and standard deviations for the two quality grading categories (Grade 1 embryos + Grade 2 embryos and Grade 1 embryos + Grade 2 embryos + Grade 3 embryos).

[0185] Table 4: Embryo evaluation (mean and standard deviation)

[0186]

[0187] The statistical analysis results of 1 grade embryo+2 grade embryo are summarized in Table 5.For (1 grade+2 grade) embryo that can be transplanted, statistically significant dose effect (Table 5) is detected.Along with the increase of dosage, we observe that the quantity of the embryo that can be transplanted increases.Based on paired comparisons, with regard to the quantity of the embryo that can be transplanted, there is no significant difference (P>0.05) between the three highest doses.In addition, for embryonic vigor, statistically significant dose effect (Table 5) is detected.

[0188] The number of ovulating cows was higher in the dose groups above 50 μg. The average ovulation rate in the higher (200 μg-400 μg) dose groups was 93%.

[0189] Table 5: Statistics of embryos that can be transferred (grade 1+grade 2) and embryo viability (least square mean, mean square error) Summary of the analysis

[0190]

[0191]

[0192] abc: Within a column, values ​​without common letters are significantly different at P < 0.05.

[0193] The statistical analysis results of 1 grade embryo+2 grade embryo+3 grade embryo are summarized in Table 6.For (1 grade+2 grade+3 grade) embryo that can be transplanted, statistically significant dose effect (Table 6) is detected.Along with the increase of dosage, the quantity of the embryo that can be transplanted increases.Based on paired comparisons, with regard to the quantity of the embryo that can be transplanted, there is no significant difference (P>0.05) between the three highest doses.In addition, for embryonic vigor, statistically significant dose effect (Table 6) is detected.

[0194] Table 6: Embryos that can be transferred (grade 1+grade 2+grade 3) and embryo viability (least square mean, mean square error) Summary of statistical analysis

[0195]

[0196] ab: Within a column, values ​​without common letters are significantly different at P < 0.05.

[0197] in conclusion

[0198] The aim of this study was to evaluate the efficacy of recombinant FSH (doses of 50 μg, 100 μg, 200 μg, 300 μg and 400 μg; n=10 / group) in domestic beef cattle. No safety issues were found in this study. RIPA follitropin alfa (bovine) stimulated follicular development in a dose-dependent manner as demonstrated by the increased number of follicles observed on day 3. This translated into a similar relationship observed for the total oocyte number, which also increased with increasing doses. The mean number of corpora lutea and the corresponding total oocyte number were consistent, indicating the efficiency of corpora lutea counts and oocyte / embryo collection. High ovulation rates were observed, except in the 50 μg group.

[0199] Evaluation is directed to the endpoint of grade 1 embryo+grade 2 embryo and grade 1 embryo+grade 2 embryo+grade 3 embryo.Grade 1 embryo and grade 2 embryo are considered to be able to be frozen and can be transplanted, while grade 3 embryo is considered to be able to be transplanted, but cannot be frozen.Regardless of whether grade 3 embryo is considered, the result is similar.The number of high-quality (grade 1 and grade 2) embryos produced by each flushing of treatment group 3, treatment group 4 and treatment group 5 (200 μ g, 300 μ g and 400 μ g, respectively) is more than the group of applying 50 μ g or 100 μ g RIPA follitropin α (cow).Therefore, 50 μ g dosage and 100 μ g dosage are invalid, while 200 μ g dosage, 300 μ g dosage and 400 μ g dosage are effective.There is no significant difference in the percentage of vitality between three effective dose groups (group 3, group 4 and group 5).In general, this study shows that the dosage between 200 μ g and 400 μ g produces the desired result, and these dosages can be used for the purpose of superovulation of domestic beef cattle.

[0200] Example 2: One dose of bovine follicle stimulating hormone induces superovulation in mature dairy cows of the domestic bovine subtype

[0201] This study was a parallel-arm, placebo-controlled study designed to measure the response of RIPA follitropin alfa (bovine) in dairy cows under field use conditions. There were two treatment groups (saline negative control and 300 μg / cow) with 20 cows in each group (groups were enrolled in cohorts until full enrollment was achieved).

[0202] Table 7: Treatment summary

[0203]

[0204] IVP

[0205] The ingredients of the formulation used were as described in Example 1.

[0206] effect

[0207] Of the 20 cows treated with 300 μg of FSH, all 20 cows produced embryos that were able to be implanted (embryo quality code 1+2+3 and developmental stage 3-9).

[0208] For the primary endpoint, the mean number of embryos that could be transferred and their respective confidence limits are shown in Table 8. The mean number of embryos that could be transferred was 7.25, and the lower limit of the 95% confidence limit was greater than 1. The mean number of embryos that could be transferred and the upper and lower 95% confidence limits indicated that 300 μg FSH was effective for superovulation of dairy cows, thereby meeting the primary efficacy criteria.

[0209] For the secondary endpoint of efficacy, the mean number of embryos that could be frozen and their respective confidence limits are shown in Table 9. The mean number of embryos that could be transferred was 6.03, and the lower limit of the 95% confidence limit was greater than 1. The mean number of embryos that could be frozen and the upper and lower 95% confidence limits indicated that 300 μg FSH was effective for superovulation of dairy cows, meeting the primary efficacy criteria for this secondary endpoint.

[0210] Table 8: Mean (standard error) of embryos harvested from cows treated with 300 μg of rbFSH that were able to be transferred There are lower and upper confidence limits

[0211]

[0212] Table 9: Mean (standard error) of embryos harvested from cows treated with 300 μg of rbFSH that could be frozen There are lower and upper confidence limits

[0213]

[0214] Follicle stimulation and ovulation after treatment were measures of FSH activity and were considered secondary endpoints.

[0215] Follicle stimulation was measured by counting the number of medium and large follicles after treatment. The results are listed in Table 10A. The average number of medium and large follicles after treatment with placebo and RIPA follitropin alpha (bovine) was 3.79 and 24.10, respectively. This shows that the ovaries of animals treated with RIPA follitropin alpha (bovine) had significantly higher stimulation compared to the ovaries from the placebo group.

[0216] Ovulation was measured by counting the number of corpora lutea on the ovaries on day 12. The results are listed in Table 10B. The average number of corpora lutea after treatment with placebo and RIPA follitropin alfa (bovine) was 0.867 and 15.00, respectively. This indicates that the number of ovulations per animal was significantly higher in the RIPA follitropin alfa (bovine) group compared to the placebo group.

[0217] Table 10A: Mean number of medium + large follicles measured on day 3 after treatment on day 0 (standard error), With lower and upper confidence limits

[0218]

[0219] Table 10B: Mean number of corpora lutea measured on day 12 after treatment on day 0 (standard error), with confidence limits Limit and upper confidence limit

[0220]

[0221] Security

[0222] The overall safety of RIPA follitropin alfa (bovine) in dairy cows was assessed by routine general health observations. No abnormal health observations were recorded in any cow during the study.

[0223] in conclusion

[0224] The results of this study suggest that a 300 μg dose of FSH is safe and effective when administered to dairy cows of the domestic subtype for superovulation.

[0225] Example 3: Improved Methods for Oocyte Collection in Bovine Cows

[0226] This example reports a study of the efficacy of FSH on optimized oocyte recovery in domestic bovine cows. The study was designed to test the response to single-chain recombinant FSH (RIPA follitropin alpha (bovine)) under field use conditions to determine if and how single-chain FSH could improve oocyte recovery.

[0227] Three treatment groups (50 μg / cow, 100 μg / cow and 200 μg / cow) were used with 20 cows in each group, summarized as follows:

[0228] Table 11: Treatment summary

[0229]

[0230]

[0231] Reproductively mature domestic beef cattle (e.g., Angus and Angus cross) at least 13 months old were used in this study. Cows with normal physical examination, confirmed to be in estrus from day 7 to day 13, non-pregnant, and with normal reproductive cycles from day 1 were enrolled.

[0232] Treatment and application

[0233] All 20 animals in each treatment group were paired according to treatment group and administered FSH: Group A: 50 μg / cow (1 mL RIPA follitropin alpha (bovine)), Group B: 100 μg / cow (2 mL RIPA follitropin alpha (bovine)) and Group C: 200 μg / cow (4 mL RIPA follitropin alpha (bovine)). Treatment was administered once intramuscularly in the neck using a sterile disposable syringe equipped with a sterile disposable 1.5-inch 20-gauge needle on day 0. A new needle and syringe were used to treat each animal.

[0234] Efficacy parameters

[0235] On day 3, an ultrasound examination was performed to assess the number and size of follicles on each ovary (small = <3 mm, medium = 3 mm-7 mm, and large ≥8 mm). Oocytes were then aspirated.

[0236] Oocyte recovery rate was defined as the percentage of oocytes per average-sized follicle.

[0237] For a cow to be considered a treatment success, both follicle size (≥40% medium follicles, preferably above 50%) and recovery efficiency should preferably be met.

[0238] Embryos were classified according to the following criteria:

[0239] Grade 1 - Excellent or good (symmetrical and spherical embryo masses with uniform size, color and density of individual blastomeres). Grade 1 embryos are eligible for freezing

[0240] Grade 2 - Fair quality (moderate irregularities in the overall shape of the embryonic mass or in the size, color, and density of the blastomeres). Grade 2 embryos are eligible for freezing

[0241] .Grade 3 – Lower quality (irregularities in the shape of the embryonic mass or irregularities in the size, color and density of the blastomeres). Grade 3 embryos are suitable for transfer but cannot be frozen.

[0242] result

[0243] The follicles produced

[0244] The table below summarizes the % large vs. small vs. medium follicles in the treated animals.

[0245] TRT: Treatment group

[0246] LSM: Least Squares Mean

[0247] SE: Standard error

[0248] Table 12: Percentage of small follicles

[0249]

[0250] Table 13: Percentage of medium follicles

[0251]

[0252] Table 14: Percentage of large follicles

[0253]

[0254] It is noteworthy that treatment groups B and C had a high percentage of large follicles compared to treatment group A.

[0255] Treatment A had a significantly higher percentage of small follicles. In contrast, treatments B and C had far fewer small follicles.

[0256] Cows treated with Treatment A had the lowest percentage of medium sized follicles. Cows in Treatments B and C both had over 50% of medium sized follicles.

[0257] These data show that Treatments B and C induced follicles with the most efficient size and contour.

[0258] Table 15: Recovery Factor

[0259]

[0260] These results show a very effective recovery rate. The recovery rate was particularly effective in Group B, which combined with the follicle profile provided a highly effective treatment in the OPU protocol.

[0261] Table 16: Embryo development rate

[0262]

[0263] These data show that the collected oocytes have a very high yield in subsequent embryonic development.

[0264] This example demonstrates that a single injection of 100 μg or 200 μg FSH can achieve optimal OPU. Considering oocyte recovery rate, 100 μg even appears to be the most effective.

[0265] Example 4: Improved Methods for Ovum Collection in Bovine Heifers

[0266] A study was conducted to evaluate the effect of treatment with long-acting recombinant FSH on OPU efficacy in Holstein heifers. The effect of treatment on follicle diameter, viable oocyte rate, and blastocyst rate was evaluated. Ninety Holstein heifers were used. On any day of the estrous cycle, heifers received an intravaginal progesterone device (PRID) and 2 mg of estradiol benzoate plus IM (day 0). Heifers in the control group received no further treatment, while heifers in the r-FSH group received a single dose of 50 μg or 100 μg of rFSH on day 4. On day 7, PRID was removed and OPU was performed in all groups. The same operator aspirated all donors, and the same batch of sex-controlled semen was used for in vitro embryo generation in all groups.

[0267] Figure 2 The number of small, medium and large follicles depending on the dose of rFSH administered is shown. There was no difference between the groups in the total number of follicles aspirated during each OPU (control = 13.2 ± 1.4 vs. 50 μg = 11.9 ± 1.2 vs. 100 μg = 11.6 ± 0.9; P = 0.90). However, when compared to the control group, regardless of the dose (50 μg or 100 μg), treatment with r-FSH reduced the number of small follicles (<6 mm) and increased the number of medium follicles (6 mm-10 mm). Heifers receiving 100 μg of r-FSH had a greater number of large follicles (<10 mm) than controls at OPU.

[0268] Table 17: OPU response of heifer donors (Holstein cows) subjected to different doses of rFSH treatment prior to OPU Summary (mean ± SEM) .

[0269]

[0270] The viable oocyte rate of heifers treated with 100 μg of rFSH was higher (P=0.004) than that of the control group (control=54.0% b For 50 μg = 62.0% ab For 100 μg = 71.0% a ; P = 0.004), without affecting the recovery factor.

[0271] In addition, heifers treated with 100 μg of rFSH had a higher cleavage rate (control = 30.9%). b For 50 μg = 41.4% ab For 100 μg = 52.3% a ; P = 0.02) and blastocyst rate (control = 7.1% b For 50 μg = 16.2% ab For 100 μg = 17.4% a ; P = 0.05).

[0272] In conclusion, treatment with a single dose of r-FSH improved the efficiency of OPU in Holstein heifers. The treatment increased the number of medium and large follicles at the time of aspiration without affecting the recovery rate. In addition, the treatment increased the viable oocyte rate, cleavage rate, and blastocyst rate.

[0273] Example 5: Improved Method for Oocyte Collection in Lactating Bovine Cows

[0274] A study was conducted to evaluate the effect of treatment with long-acting recombinant FSH on the efficacy of OPU in lactating Holstein cow donors. The effect of treatment on follicle diameter, viable oocyte rate, and blastocyst rate at OPU was evaluated. One hundred and twenty-two donors were used. On any day of the estrous cycle, cows received an intravaginal progesterone device (PRID) and 2 mg of estradiol benzoate plus IM (day 0). Cows in the control group (n=31) received no further treatment, while cows in the r-FSH group received a single dose of 100 μg (n=31) or 150 μg (n=33) of r-FSH on day 4. Cows in the p-FSH group (Foiltropin, Vetoquinol; n=27) received a total dose of 200 mg of p-FSH on days 4 and 5, divided into four decreasing doses with 12 hours intervals (57 mg, 57 mg, 43 mg and 43 mg). On day 7, PRID was removed and OPU was performed in all groups. The same operator aspirated all donors and the same batch of sex-controlled semen was used for in vitro embryo generation in all groups.

[0275] Figure 3 The number of small, medium and large follicles of lactating cows subjected to OPU with different doses of rFSH is shown. There was no difference between the groups (P = 0.21) in the total number of follicles aspirated during each OPU (mean value was 12.6 ± 0.98). However, r-FSH (regardless of dose; 100 μg or 150 μg) and p-FSH treatment reduced the number of small follicles (<6 mm) and increased the number of large follicles (<10 mm) at OPU compared to the control group (P < 0.0001).

[0276] Table 18: OPU response (mean) of lactating Holstein cow donors treated with different doses of rFSH before OPU Summary of mean ± SEM

[0277]

[0278]

[0279] The blastocyst rate (number of blastocysts / number of cultured oocytes) was higher in donors treated with 150 μg of r-FSH (27.4%) when compared to donors treated with 100 μg of r-FSH (14.1%; P=0.01).

[0280] Furthermore, the dose of 150 μg of r-FSH (2.58 ± 0.39) showed a higher number of blastocysts when compared with the 100 μg of r-FSH (1.58 ± 0.30) and p-FSH (1.46 ± 0.25) groups (P = 0.07).

[0281] Example 6: Effect of rFSH treatment on the quality of in vitro embryo production

[0282] The P / ET (pregnancy rate) of in vitro generated embryos from Holstein donors (heifers, lactating cows, non-lactating cows) treated with a single dose of 100 μg of r-FSH 3 days before OPU was also evaluated. The embryos were transferred into recipients (lactating Holstein cows) on the same day by the same operator.

[0283] Table 19: Pregnancy rates in recipients receiving in vitro generated embryos from Holstein cow donors

[0284]

[0285] Recipients receiving embryos from donors treated with rFSH had an increased P / ET [control: 27.1% (39 / 144) vs. r-FSH 35.1% (60 / 171); P = 0.06].

[0286] These data show that treatment with a single dose of rFSH prior to OPU not only increased the number of embryos produced per OPU per donor, but also overall improved the quality of those embryos, which translated into higher levels of pregnancy establishment and more live calves.

Claims

1. Use of follicle stimulating hormone (FSH) or a composition comprising FSH in a method for oocyte retrieval in a bovine cow, wherein the FSH is administered to the bovine cow in a dose of between 100 μg and 200 μg.

2. The use according to claim 1, wherein the FSH is administered once during the estrous cycle.

3. The use according to claim 1 or 2, wherein the FSH is administered at a dose of 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg or 200 μg, preferably at a dose between 100 μg and 150 μg, even more preferably at a dose of about 100 μg.

4. Use according to any one of the preceding claims, wherein the FSH is administered by systemic administration, preferably by injection, more preferably by intramuscular injection.

5. Use according to any one of the preceding claims, wherein the α subunit is a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 1, and / or the β subunit is a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:

2.

6. Use according to any one of the preceding claims, wherein the FSH comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, preferably at least 96%, at least 97%, at least 98% or at least 99% identical.

7. The use according to claim 6, wherein the FSH comprises, consists essentially of or consists of SEQ ID NO:

3.

8. Use according to any one of the preceding claims, wherein the bovine cow is a heifer, and wherein the FSH is administered to the bovine cow at a dose of 100 μg.

9. The use according to any one of claims 1 to 7, wherein the bovine cow is a lactating cow, and wherein the FSH is administered to the bovine cow at a dose of 150 μg.

10. A method for collecting oocytes from a bovine cow, the method comprising (i) administering FSH once to the cow at a dose of between 100 μg and 200 μg; and (ii) collecting oocytes from the cow.

11. The method for collecting oocytes according to claim 10, wherein FSH is administered during the estrous cycle.

12. The method of claim 10 or 11, wherein the bovine cow is a heifer, and wherein the FSH is administered to the bovine cow at a dose of 100 μg.

13. The method of claim 10 or 11, wherein the bovine cow is a lactating cow, and wherein the FSH is administered to the bovine cow at a dose of 150 μg.

14. Use according to any one of claims 1 to 9 or method according to any one of claims 10 to 13, wherein oocyte collection is performed 1 to 5 days, preferably 2 to 4 days, even more preferably about 3 days after FSH administration.

15. The use or method according to any one of the preceding claims, comprising: (a) providing bovine cows or groups of bovine cows; (b) administering a dose of about 100 μg of FSH to said one or more cows during the estrous cycle, preferably by injection, more preferably by intramuscular injection; and (c) collecting oocytes from the one or more cows.

16. The use or method according to any one of the preceding claims, further comprising fertilizing the oocyte in vitro.

17. Use or method according to any one of the preceding claims, wherein the FSH is formulated as a composition with one or more excipients or carriers.

18. A method for preparing a fertilized cow oocyte, wherein the method comprises: A single dose of between 100 μg and 200 μg of FSH is administered to a bovine cow during the estrous cycle, oocytes are collected from the cow, the oocytes are fertilized in vitro, and the fertilized oocytes are optionally conditioned.

19. The method of claim 18, wherein the bovine cow is a heifer, and wherein the FSH is administered to the bovine cow at a dose of 100 μg.

20. The method of claim 18, wherein the bovine cow is a lactating cow, and wherein the FSH is administered to the bovine cow at a dose of 150 μg.

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