Use of GnRH antagonists for synchronized follicular wave appearance in mammals

Abstract

The present disclosure relates to methods of reproductive management of mammals using GnRH antagonists, such as cetrorelix. In particular, the GnRH antagonists may be used to synchronize follicular wave appearance in female mammalian populations, and / or for fixed time reproductive management protocols, such as oocyte collection protocols, embryo collection protocols, artificial insemination protocols, or embryo transfer protocols. Also described are devices and kits for reproductive management comprising a GnRH antagonist and optionally one or more additional drugs useful for reproductive management.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 390,080, filed on July 18, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] The computer readable form of the sequence listing "13764-P65604PC00_SequenceListing.XML" (4,452 bytes) created on July 17, 2023 is incorporated herein by reference. Technical Field

[0005] The present disclosure generally relates to methods, uses, devices and kits for reproductive management in mammals using GnRH antagonists. Specifically, the present disclosure relates to the use of GnRH antagonists for synchronizing follicular wave emergence (FWE) in mammals. The methods, uses, devices and kits can be applied to ovulation synchronization, fixed-time artificial insemination protocols and other reproductive management strategies. Background Art

[0006] Existing animal breeding management programs for fixed-time artificial insemination and donor / recipient management (mainly cattle and sheep) for embryo transfer synchronize the emergence of new follicle waves as a basic reset point. This concept has not been clinically practiced in humans. The current method for synchronizing the emergence of ovarian follicle waves (FWE) in animals includes ultrasound-guided ablation of estradiol, gonadotropin-releasing hormone / luteinizing hormone (luteinizing hormone) (GnRH / LH), prostaglandins (PGF) or dominant follicles, or a combination of these various treatments. Estradiol is the most effective drug, and single injection treatment is widely used in South American countries, but has been banned by the European Union for reproductive management of food animals. In other parts of the world (including the United States and Canada), estradiol is not preferred due to concerns about the environmental effects of estrogen compounds. Single treatment with GnRH / LH and / or prostaglandins (as a monotherapy, or as a combination of a single GnRH / LH injection and a single prostaglandin injection) is not as effective due to reliance on inducing ovulation. Combination regimens such as GnRH + progesterone controlled internal drug release (CIDR)-PGF-GnRH (e.g., Ovsynch or co-synch regimens) and multi-dose regimens (e.g., two injections of PGF) often involve multiple treatments at 7- or 10-day intervals. Follicle ablation is the most effective method for inducing FWE, but requires expensive equipment and technical skills and is not practical at the herd level. According to conservative estimates, more than 15 million cattle undergo artificial insemination each year in Brazil alone, one of the largest markets.

[0007] Ultrasound studies in the early 1990s characterized the pattern of follicular growth in cattle, and since then the bovine model has been widely used to understand patterns in other mammalian species, including humans. Follicular growth in cattle occurs in a wave-like pattern, with most estrous cycles consisting of two to three waves. In cattle, each follicular wave is characterized by the growth of 8 to 41 small follicles (2-4 mm in diameter). The appearance of each wave is preceded by a surge in follicle-stimulating hormone (FSH). At the beginning of a wave, all follicles require FSH for growth; therefore, the appearance of follicular waves in cattle and other mammals is temporally correlated with circulating FSH levels. Bovine follicles grow at approximately the same rate for approximately 2 days, after which one follicle is “selected” to become the dominant follicle, while the remaining follicles stop growing and degenerate (Ginther et al., 1989; Knopf et al., 1989). Due to feedback from follicular products to the hypothalamus and pituitary (Adams and Singh, 2021), the dominant follicle (size ) is associated with a decline in circulating FSH levels (Adams et al., 1992). All follicles in a wave have growth (increasing diameter), resting (constant diameter), and degeneration (decreasing diameter) phases. The presumed dominant follicle differentially produces luteinizing hormone (LH) receptor expression close to the time of selection (3 days after the emergence of the wave in cattle), while the subordinate follicles have no LH receptors or have low LH receptors. FSH plasma levels decrease between the time of wave emergence and the selection of the dominant follicle. Studies have shown that the granulosa cells of the dominant follicles in cattle acquire LH receptors at about the time of selection, so that the dominant follicles continue to grow with LH support in the face of the FSH nadir (Adams and Singh, 2021). The dominant follicle produces substances (mainly estradiol) that keep FSH low and prevent the next wave from appearing. After the dominant follicle degenerates or ovulates, follicular products (such as estradiol) decrease to a sufficiently low level to allow a surge in circulating concentrations of FSH, leading to the emergence of a new wave.

[0008] Cattle and camelids release one egg / oocyte at ovulation (i.e., both are monozygotic mammals) and give birth to a single offspring. Sheep and goats are polyzygotic species, giving birth to 2 to 3 lambs / lambs. The control mechanisms of wave emergence and follicle growth are similar between cows, sheep, goats and camelids. However, unlike other ruminant species, camelids are classified as induced ovulators, in which mating stimulation is necessary for ovulation to occur (San Martin et al., 1968; England et al., 1969; Fernandez-Baca et al., 1970a). The presence of functional dominant follicles plays a key role in the ability of camelids to accept mating and ovulation (Adams et al., 1990; Bravo et al., 1991; Vaughan et al., 2004). If mating (and subsequent ovulation) does not occur, development of the dominant follicle continues in a wave-like pattern characterized by a periodic increase in the number of follicles every 20 to 25 days (interwave interval) and selection of the dominant follicle, followed by regression (Adams et al., 1990; Vaughan et al., 2004). To simplify herd management practices, ovulation of a group of llamas can be synchronized for artificial insemination by inducing a new wave of follicles. Hormonal studies in camelids have attempted to use GnRH (Bravo et al., 1992; Aller et al., 1999; Cancino et al., 1999; Huanca et al., 2001), LH (Huanca et al., 2001; Taylor et al., 2000) and hCG (Adam et al., 1992; Bourke et al., 1992; Correa et al., 1997; Ratto et al., 1997) to induce ovulation, however, results remain variable.

[0009] Both the selection of the dominant follicle and ovulation require hormonal stimulation from FSH and LH (Adams et al., 2008). After GnRH is secreted from the hypothalamus, both FSH and LH are released from the anterior pituitary. In human assisted reproductive practice, endogenous GnRH membrane receptors (located in the pituitary) are usually inhibited by GnRH antagonists (such as Cetrorelix) (Kovacs et al., 2001). Cetrorelix (trade name: Cetrotide) TM ) has been used as a fertility treatment for women to avoid a premature increase in LH during ovarian stimulation (EMD Serono Inc., 2008). Its effect of preventing existing follicles from ovulating is well known and is therefore used clinically in human FSH stimulation protocols for assisted reproduction. In such protocols, Cetrorelix is ​​administered simultaneously with FSH to block ovulation of FSH-induced follicles.

[0010] Cetrorelix is ​​a synthetic decapeptide that acts as a GnRH / LHRH antagonist (acetyl-D-3-(2′-naphthyl)-alanine-D-4-chlorophenylalanine-D-3-(3′-pyridinyl)-alanine-L-serine-L-tyrosine-D-citrulline-L-leucine-L-arginine-L-proline-D-alanine-amide; SEQ ID NO: 2). Cetrorelix occupies the GnRH receptors on gonadotropes in the pituitary, thereby preventing the release of LH even in the presence of GnRH pulses. In human assisted reproduction protocols, Cetrorelix is ​​used as daily subcutaneous (s / c) injections of 0.25 mg for up to 6 days, or as an initial s / c bolus of 3 mg followed by a daily dose of 0.25 mg after the first 4 days. Its elimination half-life is quite short (5 hr after a single injection of 0.25 mg in women).

[0011] Cetrorelix is ​​known to reduce the circulating concentration of LH, inhibit the growth of existing single dominant follicles or multiple large follicles (e.g., during FSH stimulation of the ovaries) and prevent premature ovulation. Cetrorelix is ​​clinically used in women's assisted reproductive technology (e.g., preventing premature ovulation during FSH stimulation in human IVF cycles). Other GnRH antagonists are also used for this purpose in human assisted reproduction. During the human FSH stimulation regimen, multiple follicles grow to ovulation size, and Cetrorelix is ​​co-administered with FSH to prevent ovulation of existing multiple dominant follicles during treatment.

[0012] The effects of GnRH antagonists such as cetrorelix have been studied in cattle and other domestic animals.

[0013] Nivet et al. (2018) investigated the mechanisms behind oocyte competence when LH was suppressed by cetrorelix. Cetrorelix was given to heifers for 4 days at the end of superstimulation. A significant reduction in the number of follicles >10 mm in diameter was observed during the dominant stage of development.

[0014] Haughian et al. (2013) described the effects of the GnRH antagonist acyline on follicular development. Acyline was given just before ovulation and multiple injections continued after ovulation and the appearance of the follicular wave. Acyline did not reduce FSH concentrations during the periovulatory surge, and early follicular development was not affected, although subsequent growth of the dominant follicle (>8.0 mm) was arrested by acyline. Failure to select a dominant follicle in the acyline group was associated with a decrease in the concentration of LH rather than FSH. The maximum diameter of the dominant follicle in control animals (13.3 mm) was larger than that in acyline-treated animals (7.7 mm).

[0015] Ginther et al. (2012) described the effects of acyline on follicular development. Acyline was given to heifers on days 15, 16, and 17 after ovulation. It caused regression of the dominant follicles of wave 2 and the emergence of wave 3. The interovulation intervals in heifers treated with acyline ranged from 25 to 34 days compared to 19 to 23 days in untreated heifers. Synchronization of wave emergence was not examined or discussed.

[0016] Silva et al. (2011) described the effects of cetrorelix on llama follicular development. Cetrorelix inhibited the preovulatory LH surge and ovulation induced by GnRH and ovulation inhibitory factor (OIF) in llamas.

[0017] Ulker et al. (2001) reported that cetrorelix inhibited LH and progesterone secretion in cattle without affecting the length of the estrous cycle.

[0018] Oussaid et al. (2000) described the effects of the GnRH antagonist antarelix on follicular development. Treatment of superstimulated heifers with antarelix for 36-60 hours resulted in follicular atresia without affecting oocyte competence as determined by in vitro embryo production.

[0019] Fike et al. (1997) described the effects of the LHRH antagonist SB-75 on follicular development. Treatment of animals with SB-75 from day 2 to day 12 of the estrous cycle resulted in modulation of circulating LH and FSH and inhibition of dominant follicle persistence and estradiol secretion. The effects of SB-75 on dominant follicle size and follicle number were described.

[0020] Madill et al. (1994) described the effects of SB-75 on hormone levels and ovulation. SB-75 can be used to delay the LH surge (detected by radioimmunoassay analysis of blood samples) and ovulation (detected by ultrasound) in superovulated heifers.

[0021] Leonardi (2018) experimentally used GnRH antagonists in cattle to test the effects of kisspeptin. The study involved the administration of cetrorelix on day 6 of follicular development when plasma progesterone concentrations are low. Summary of the invention

[0022] There is a need to develop new methods for manipulating follicular waves in large and small ruminants, camelids, and other livestock that are both effective and efficient when implemented under field conditions. As described herein, the inventors have demonstrated that administration of the GnRH antagonist cetrorelix results in the appearance of new follicular waves in a consistent time frame in female mammals, such as cattle and alpacas. Thus, the present disclosure relates to the use of GnRH antagonists for reproductive management in female mammals. The methods and uses described herein are non-therapeutic.

[0023] One aspect includes a non-therapeutic method of synchronizing follicular wave emergence (FWE) in a population of female mammals, the method comprising administering an effective amount of a GnRH antagonist to each mammal in the population of female mammals.

[0024] In one embodiment, GnRH antagonists include cetrorelix, acyline, teverelix, degarelix, ganirelix, antide, relμgolix, elagolix, abarelix, prazarelix, ramorelix, antide, detirelix, ozarelix, linzagolix, opigolix, sufμgolix, and A-75998. In one embodiment, GnRH antagonists include abarelix. In one embodiment, GnRH antagonists include degarelix. In one embodiment, GnRH antagonists include relμgolix. In one embodiment, GnRH antagonists include cetrorelix.

[0025] In one embodiment, the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

[0026] In one embodiment, administration of the GnRH antagonist comprises at least one treatment, optionally 2 or 3 treatments.

[0027] In one embodiment, the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of about 2.5 μg / kg body weight to about 40 μg / kg body weight, optionally at a dose of about 5 μg / kg, 7.5 μg / kg, 10 μg / kg or 20 μg / kg body weight.

[0028] In one embodiment, the GnRH antagonist is administered via a vaginal drug release device.

[0029] In one embodiment, the method further comprises co-administering a progesterone or a progesterone analog, optionally by a vaginal drug release device.

[0030] In one embodiment, the mammal is livestock, optionally the livestock is a bovine, camelid, equine, porcine, ovine or caprine species.

[0031] In one embodiment, the mammal is a pet or wild animal, optionally a bison, elk, reindeer, deer, musk ox, non-human primate, canine, or feline, optionally a tiger, lion, or panther.

[0032] In one embodiment, the mammal is a human.

[0033] In one embodiment, the population is a mixed population.

[0034] In one embodiment, the method further comprises inducing ovulation in each mammal in the population of female mammals at a fixed time after administration of the GnRH antagonist, comprising

[0035] 1) administering an effective amount of an ovulation inducing agent at a fixed time after administration of a GnRH antagonist; or

[0036] II) co-administering an effective amount of progesterone or a progesterone analog with a GnRH antagonist; discontinuing the progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; and administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the progesterone or a progesterone analog and / or administering PGF or a PGF analog.

[0037] In one embodiment, the method further comprises artificially inseminating each mammal in the population of female mammals at a fixed time after administration of the ovulation inducing agent.

[0038] In one embodiment, the method further comprises mating each mammal in the population of female mammals at a fixed time after administration of the ovulation inducing agent.

[0039] In one embodiment, the method further comprises transferring the embryo to each animal in the population of female mammals at a fixed time after administration of the ovulation inducing agent.

[0040] Another aspect includes a non-therapeutic method of synchronizing ovulation in a population of female mammals, the method comprising:

[0041] I) a) administering an effective amount of a GnRH antagonist to each mammal in a population of female mammals; and b) administering an effective amount of an ovulation inducing agent to each mammal in a population of female mammals at a fixed time after the administration of the GnRH antagonist; or

[0042] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to each mammal in a population of female mammals; b) discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; and c) administering an effective amount of an ovulation inducing agent to each mammal in a population of female mammals at a fixed time after discontinuing the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog.

[0043] Another aspect includes a non-therapeutic method of inducing the appearance of follicular waves in a female mammal, the method comprising: administering to the female mammal an effective amount of a GnRH antagonist.

[0044] Another aspect includes a non-therapeutic method of inducing ovulation in a female mammal, the method comprising:

[0045] I) a) administering an effective amount of a GnRH antagonist to a female mammal; and b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; or

[0046] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; and c) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog.

[0047] Another aspect includes a non-therapeutic fixed-time embryo transfer method comprising:

[0048] I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; and c) transferring one or more embryos to the female mammal at a fixed time after the administration of the ovulation inducing agent; or

[0049] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog; and d) transferring one or more embryos to the female mammal at a fixed time after the administration of the ovulation inducing agent.

[0050] Another aspect includes a non-therapeutic fixed-time superstimulation or superovulation method comprising:

[0051] I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of FSH or a FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; and c) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of FSH or a FSH analog; or

[0052] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) administering an effective amount of FSH or a FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) discontinuing the use of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the FSH or FSH agonist; and d) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the use of progesterone or a progesterone analog and / or administering PGF or a PGF analog.

[0053] In one embodiment, the method further comprises collecting one or more oocytes at a fixed time after administering an ovulation inducing agent. In one embodiment, the method further comprises I) d) or II) e) artificially inseminating a female mammal at a fixed time after administering an ovulation inducing agent; and I) e) or II) f) collecting one or more embryos at a fixed time after artificial insemination. In one embodiment, the method further comprises I) d) or II) e) mating a female mammal at a fixed time after administering an ovulation inducing agent; and I) e) or II) f) collecting one or more embryos at a fixed time after artificial insemination.

[0054] Another aspect includes a non-therapeutic fixed-time embryo collection method comprising:

[0055] I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of FSH or an FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of FSH or an FSH analog; d) artificially inseminating or mating the female mammal at a fixed time after the administration of the ovulation inducing agent; and e) collecting one or more embryos at a fixed time after the artificial insemination or mating; or

[0056] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) administering an effective amount of FSH or an FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) discontinuing the use of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the FSH or FSH agonist; d) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the use of progesterone or a progesterone analog and / or administering PGF or a PGF analog; e) artificially inseminating or mating the female mammal at a fixed time after the administration of the ovulation inducing agent; and f) collecting one or more embryos at a fixed time after the artificial insemination or mating.

[0057] Another aspect includes a non-therapeutic fixed-time oocyte collection method comprising:

[0058] I) a) administering an effective amount of a GnRH antagonist, and b) collecting one or more oocytes at a fixed time after the administration of the GnRH antagonist;

[0059] II) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of FSH or a FSH agonist to the female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; and c) collecting one or more oocytes at a fixed time after the administration of the FSH or FSH agonist;

[0060] III) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of FSH or a FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of FSH or a FSH analog; and d) collecting one or more oocytes at a fixed time after the administration of the ovulation inducing agent; or

[0061] IV) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) administering an effective amount of FSH or a FSH agonist to a female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) discontinuing the progesterone or progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the FSH or FSH agonist; d) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the progesterone or progesterone analog and / or administering the PGF or a PGF analog; and e) collecting one or more oocytes at a fixed time after the administration of the ovulation inducing agent.

[0062] Another aspect includes a non-therapeutic fixed-time artificial insemination method, the method comprising:

[0063] I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; and c) artificially inseminating the female mammal at a fixed time after the administration of the ovulation inducing agent; or

[0064] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) ceasing the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after ceasing the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog; and d) artificially inseminating the female mammal at a fixed time after the administration of the ovulation inducing agent.

[0065] Another aspect includes a breeding management method comprising:

[0066] I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; and c) mating the female mammal at a fixed time after the administration of the ovulation inducing agent; or

[0067] II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuing the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog; and d) mating the female mammal at a fixed time after the administration of the ovulation inducing agent.

[0068] In one embodiment of the aspects described herein, the GnRH antagonist comprises cetrorelix, abarelix, degarelix, ganirelix, anrelix / teverelix, prarelix, ramorelix, antitide, detirelix, ozarelix, acyline, elagolix, linzagolix, relugolix, opigolix, sufgolix, or A-75998. In one embodiment, the GnRH antagonist comprises abarelix. In one embodiment, the GnRH antagonist comprises degarelix. In one embodiment, the GnRH antagonist comprises relugolix. In one embodiment, the GnRH antagonist comprises cetrorelix.

[0069] In one embodiment of the aspects described herein, the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

[0070] In one embodiment of the aspects described herein, administering the GnRH antagonist comprises at least one treatment, optionally 2 or 3 treatments.

[0071] In one embodiment of the aspects described herein, the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of about 5 μg / kg body weight to about 40 μg / kg body weight, optionally at a dose of about 20 μg / kg body weight.

[0072] In one embodiment of the aspects described herein, the GnRH antagonist is administered via a vaginal drug release device.

[0073] In one embodiment of the aspects described herein, the mammal is livestock, optionally the livestock is bovine, camelid, equine, porcinoid, ovine or caprine. In one embodiment, the mammal is cattle.

[0074] In one embodiment of the aspects described herein, the mammal is a wild animal, optionally a bison, elk, reindeer, deer, musk ox, non-human primate, canine or feline, optionally a tiger, lion or panther.

[0075] In one embodiment of the aspects described herein, the mammal is a human.

[0076] In one embodiment of the aspects described herein, the method further comprises co-administering a progesterone, optionally wherein the progesterone is administered via a vaginal drug release device. In one embodiment, the progesterone drug release is administered for 7-8 days.

[0077] In one embodiment of the aspects described herein, the GnRH antagonist is administered on day 0 (D0), and the method further comprises inserting a progesterone drug releasing device on D0; administering an effective amount of PGF or a PGF analog and removing the progesterone drug releasing device on day 8 (D8); and administering an effective amount of GnRH on day 10 (D10).

[0078] In one embodiment of the aspects described herein, PGF or a PGF analog comprises prostaglandin 2 alpha (PGF2a), cloprostenol, dinoprost, dinoprost tromethamine, bimatoprost, travoprost, carboprost, and latanoprost. In one embodiment, PGF or a PGF analog comprises PGF2a.

[0079] In one embodiment of the aspects described herein, the ovulation induction agent includes GnRH, LH or estradiol, or any agonist, analog, ester, conjugate or recombinant product thereof. In one embodiment, GnRH or GnRH analog or agonist includes GnRH, buserelin, deslorelin, fertirelin, gonadorelin, goserelin, leuprorelin or triptorelin. In one embodiment, LH or LH analog, agonist, conjugate or recombinant product includes LH, human chorionic gonadotropin / hCG or equine chorionic gonadotropin / eCG. In one embodiment, estradiol or estradiol ester, analog or agonist includes estradiol-17β, estradiol benzoate, estradiol valerate or estradiol cypionate.

[0080] Another aspect includes a drug release device comprising a GnRH antagonist. In one embodiment, the GnRH antagonist comprises cetrorelix, abarelix, degarelix, ganirelix, anrelix / teverelix, prarelix, ramorelix, antitide, detirelix, ozarelix, acyline, elagolix, linzagolix, relugolix, opigolix, sufgolix, or A-75998. In one embodiment, the GnRH antagonist comprises abarelix. In one embodiment, the GnRH antagonist comprises degarelix. In one embodiment, the GnRH antagonist comprises relugolix. In one embodiment, the GnRH antagonist is cetrorelix.

[0081] In one embodiment, the drug release device provides controlled release of the GnRH antagonist over a period of about one day to about two days.

[0082] In one embodiment, the drug release device further comprises a progesterone, wherein the drug release device provides controlled release of the progesterone over a period of at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or longer than 10 days.

[0083] In one embodiment, the drug release device is used to synchronize follicular wave emergence (FWE) in a population of female mammals, to synchronize ovulation in a population of female mammals, to use in a fixed-time superstimulation or superovulation method, to use in a fixed-time embryo transfer method, to use in a fixed-time artificial insemination method, or to use in a fixed-time breeding method.

[0084] One aspect includes a non-therapeutic method of synchronizing the occurrence of follicular waves in a population of female mammals, the method comprising inserting a drug release device comprising a GnRH antagonist as described herein into each mammal in the population of mammals.

[0085] The foregoing sections are provided as examples only and are not intended to limit the scope of the present disclosure and the appended claims. Based on the claims, descriptions, and examples of the present invention, those of ordinary skill in the art will appreciate the additional objects and advantages associated with the compositions and methods of the present disclosure. For example, various aspects and embodiments of the present disclosure may be utilized in many combinations, all of which are expressly covered by this specification. These additional advantages, objects, and embodiments are expressly included within the scope of the present disclosure. Publications and other materials used herein to illustrate the background of the present disclosure and to provide additional details about practice in certain circumstances are incorporated by reference and, for convenience, are listed in the attached reference section.

[0086] Brief description of the attached figure

[0087] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings which show illustrative embodiments of the present disclosure, in which:

[0088] Figure 1 Schematic diagram showing the experimental design used in Example 1. KP = Kissin; Cetro = Cetrorelix.

[0089] Figure 2Follicular dynamics and LH curves in heifers treated in Example 1 are shown. A) Growth curve of dominant follicles in pubertal heifers treated with three injections of 15 mg human Kissin-10 given at 60-minute intervals over a 2-hour period on day 6 of the follicular wave (wave onset day = day 0) (Kp10 group, n=5). The Cetrorelix group (n=5) was injected with a GnRH antagonist (Cetrelix, 20 μg / Kg body weight, intramuscularly) 3 hours before Kissin treatment, while the control group (n=5) was given three intravenous injections of saline. B) Plasma LH plasma concentrations in Kp10, Cetrorelix and control group heifers.

[0090] Figure 3 Schematic diagram showing the experimental design used in Example 2. WE = Wave Emergence; Cetro = Cetrorelix.

[0091] Figure 4 A schematic timeline is shown for Example 4. Cetrorelix (1.5 mg, 3 mL of 0.5 mg / mL in 5% D-mannitol) was given to each animal daily i / m on days 1-2, 3-4 or 6-7, while the first group served as untreated controls (n=8 cows per group). PGF=prostaglandin analog, Ov ovulation, WE=wave emergence=day 0.

[0092] Figure 5 Shows the dominant follicle growth pattern after cetrorelix treatment. Cetro 1 = Day 1-2 group, Cetro 3 = Day 3-4 group, Cetro 6 = Day 6-7 group.

[0093] Figure 6 Schematic diagram showing the experimental design for Example 5.

[0094] Figure 7 Showing the dominant follicle growth pattern after a single cetrorelix treatment.

[0095] Figure 8 Schematic timeline showing the experimental design for Example 6. Ov = ovulation, Cetro = cetrorelix, hCG = human chorionic gonadotropin (Chorulon), D = day, Cuemate = intravaginal progesterone device, P4 = progesterone, LH = luteinizing hormone, PGF = prostaglandin.

[0096] Fig. 9 Schematic diagram showing the experimental design used in Example 7. PGF = prostaglandin F2α; AI = artificial insemination.

[0097] Fig.10The percentage of heifers showing estrus behavior / estrus detection (left graph) and time of ovulation after CIDR removal / PGF injection (right graph) are shown. D10, D11 and D12 refer to days 10, 11 and 12 after cetrorelix treatment, respectively. 48H, 72H and 96H refer to 48 hours, 72 hours and 96 hours after CIDR removal / PGF treatment in both panels.

[0098] Fig.11 Schematic diagram showing the experimental design used in Example 8. US = ultrasound; PGF = prostaglandin F2α; ECG = equine chorionic gonadotropin; AI = artificial insemination.

[0099] Fig.12 Shown are the levels of LH secreted by pituitary cells after addition of 1 nM GnRH (mIU / mL). Cells were pretreated with or without (control) cetrorelix, degarelix, relugolix and abarelix.

[0100] Fig.13 Shown are the levels of LH secreted by pituitary cells after addition of 10 nM GnRH (mIU / mL). Cells were pretreated with or without (control) cetrorelix, degarelix, relugolix, and abarelix.

[0101] Fig.14 Plasma LH levels after GnRH challenge in cattle pretreated with GnRH antagonists are shown. (A) Plasma LH levels in cattle after cetrorelix injection are shown (+SEM). GnRH injection was given at time 0 min: positive control (n=2) showed a 1.5-fold increase in plasma LH concentration within 30 minutes, while LH levels remained unchanged (indicated by *) in heifers pretreated with cetrorelix 60 minutes before GnRH injection (n=2). (B) Fold change in plasma LH in animals pretreated with abarelix, degarelix, relugolix and cetrorelix or controls (no pretreatment) is shown. Time 0 min = time of GnRH injection.

[0102] Fig.15 Schematic diagram showing the experimental design used in Example 11. PGF = prostaglandin F2α; FA = follicle ablation.

[0103] Fig.16 Schematic diagram showing the experimental design used in Example 12. PGF = prostaglandin F2α; FA = follicle ablation.

[0104] Fig.17 Schematic diagram showing the experimental design used in Example 13. PGF = prostaglandin F2α; FA = follicle ablation.

[0105] Fig.18Schematic diagram showing the experimental design used in Example 14. PGF = Prostaglandin F2α; AI = Artificial Insemination; OV = Ovulation; Preg test = Pregnancy test; US = Ultrasound. DETAILED DESCRIPTION

[0106] The following is a detailed description provided to help those skilled in the art practice the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. The terms used in the description herein are only used to describe specific embodiments and are not intended to limit the present disclosure. All publications, patent applications, patents, drawings and other references mentioned herein are expressly incorporated by reference in their entirety.

[0107] In addition, as will be appreciated by those skilled in the art, the definitions and embodiments described in a particular section are intended to apply to other embodiments described herein to which they are suitable. For example, in the following paragraphs, different aspects of the present disclosure are defined in more detail. Unless expressly indicated to the contrary, each aspect so defined may be combined with any other one or more aspects. In particular, any feature described herein may be combined with any other one or more features described herein.

[0108] The present disclosure relates to non-therapeutic uses of GnRH antagonists for reproductive management of mammals. As described herein, the inventors have confirmed that the administration of the GnRH antagonist cetrorelix causes the synchronous appearance of new follicular waves in female mammals (such as cattle and alpacas). For example, a single cetrorelix injection of 1.5 mg in an asynchronous alpaca population resulted in the appearance of new follicular waves after an average of 5.33 days (with a variance of 7.38), compared to an average of 8.93 days (with a variance of 22.067) in control animals. The lower variance observed in the cetrorelix treatment group (7.38 to 22.067) indicates that synchronous FWE relative to the control occurs. Since the mechanisms controlling follicular dynamics are similar in all mammals, it is expected that this synchronous FWE after the administration of a GnRH antagonist such as cetrorelix will be reproduced in other mammalian species, albeit with species-specific temporal dynamics. Thus, cetrorelix (and related GnRH receptor antagonists) can be used to manipulate the appearance of the next follicular wave in female mammals, including, for example, cattle, other domestic animals, wild mammals, non-human primates, and humans.The methods and uses described herein are non-therapeutic.

[0109] It is known that Cetrorelix is ​​used in people to prevent premature ovulation of multiple existing dominant follicles in FSH stimulation IVF cycles. In contrast, the method described herein allows the use of a single injection or intravaginal application of a GnRH antagonist at any stage of the ovarian cycle (e.g., as part of an intravaginal drug release device, optionally in combination with progesterone) to reset the appearance of the next (new) follicle wave at a consistent time from the treatment time. Without wishing to be limited by theory, Cetrorelix seems to rely on the fact that LH is consistently reduced in all animals at the same time to synchronize the appearance of follicle waves. It may involve endocrine pathways that are identical to the effect of estradiol on the appearance of waves but are at different levels (estradiol prevents GnRH from releasing, and Cetrorelix blocks LH from releasing). This "synchronization of ovarian follicle waves" is the basis for ovulation synchronization and fixed-time artificial insemination schemes in cattle and other domestic animals, as well as gonadotropin stimulation and superovulation treatments for embryo production / transfer.

[0110] A single injection of cetrorelix or other GnRH antagonist compound is an alternative to the use of estradiol and / or GnRH for ovarian follicular wave synchronization in mammals, including humans, because it is nonsteroidal, easily and inexpensively synthesized, effective for all stages of the dominant follicle, and requires only a single treatment. In addition, cetrorelix or other GnRH antagonists can be impregnated as a rapid-release drug in silicone or similar compounds to prepare an intravaginal drug release device, optionally in combination with a progesterone, which would simplify handling (single device rather than injections) and prevent secondary use of previously used devices.

[0111] I. Definitions

[0112] As used herein, unless otherwise indicated, the following terms may have the meanings assigned to them below. However, it should be understood that other meanings known or understood by those of ordinary skill in the art are also possible and within the scope of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.

[0113] When providing a range of values, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly specifies otherwise, to 1 / 10 of the lower limit unit) and any other described value or intermediate value range in the described range are included in this specification. Smaller ranges from any lower limit to any upper limit are included. The upper and lower limits of these smaller ranges that can be independently included in the smaller ranges are also included in this specification, subject to any explicitly excluded limits in the described range. In the case where the described range includes one or two of the limits, the range excluding any one or two of those included limits is also included in this specification.

[0114] All numerical values ​​in the detailed description and claims herein are modified by the indicated value of "about" or "approximately", and take into account experimental errors and variations that would be expected by a person of ordinary skill in the art. For example, "about" may mean plus or minus 10%, or plus or minus 5% of the indicated value mentioned.

[0115] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0116] As used herein, the phrase "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present in conjunction in some cases and separate in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified.

[0117] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of a plurality of elements or a list of elements, but also including more than one, and optionally, additional unlisted items. Only when the opposite term is clearly indicated, such as "only one" or "exactly one", or when used in a claim, "consisting of..." refers to including exactly one element in a plurality of elements or a list of elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (i.e., "one or the other but not two") only when there is an exclusive term (such as "any one", "one of", "only one of" or "exactly one of") in front.

[0118] As used herein, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "having," "consisting of," etc., shall be understood as open ended, i.e., meaning including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0119] As used herein, with respect to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed within the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified within the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified.

[0120] It should also be understood that in certain methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described unless the context dictates otherwise.

[0121] II. Methods and Uses

[0122] As shown herein, administration of the GnRH antagonist cetrorelix at different stages of the ovarian cycle results in the appearance of new follicular waves at a time consistent with the time of administration. Therefore, GnRH antagonists (e.g., cetrorelix) can be used to synchronize the appearance of new follicular waves and optional subsequent ovulation in a mammalian population (e.g., a livestock population). This synchronized FWE can be used for reproductive management in livestock, for example, for inducing synchronized ovulation for fixed-time oocyte collection schemes, artificial insemination schemes, breeding, and / or embryo production / transfer. Therefore, one aspect of the present disclosure is a non-therapeutic method for synchronizing follicular wave appearance (FWE) in a female mammal population, the method comprising: administering an effective amount of a GnRH antagonist to each mammal in a female mammal population. Another aspect of the present disclosure includes a non-therapeutic method for synchronizing ovulation in a female mammal population, the method comprising: administering an effective amount of a GnRH antagonist to each mammal in a female mammal population; and administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist. As will be understood in the art, in some cases, such as in species of spontaneously ovulating animals (e.g., bovine, ovine, equine, porcine, canine), individual mammals in the herd may have existing corpora lutea that may interfere with synchronization. Therefore, to avoid the need to examine individual animals for the presence of existing functional corpora lutea, additional treatments may be administered. For example, progesterone may be co-administered with a GnRH antagonist to a) prevent the LH surge and ovulation, b) prime the endometrium to prevent early PGF release after ovulation, and c) establish the ovulatory cascade with progesterone discontinuation. PGF may be administered at about the time progesterone is discontinued to induce luteolysis of any existing corpora lutea. Therefore, in one aspect, a non-therapeutic method for synchronizing ovulation in a mammal population comprises: administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to each mammal in a female mammal population; stopping administration of progesterone or a progesterone analog at a fixed time after administration of the GnRH antagonist and administering an effective amount of a prostaglandin (PGF) or a PGF analog; and administering an effective amount of an ovulation inducing agent to each mammal in a female mammal population at a fixed time after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog. The methods and uses described herein are non-therapeutic.

[0123] The GnRH antagonist may be administered at any stage of the ovulatory cycle. Thus, in various aspects and embodiments of the methods described herein, the cycle stage of the female mammals need not be known, and / or the population may be a mixed population.

[0124] In various embodiments of the aspects described herein, the method further comprises collecting one or more oocytes from each mammal at a fixed time after administration of the ovulation inducing agent, artificially inseminating each mammal, mating each mammal, collecting one or more embryos from each mammal, or transferring the embryos to each animal in the population of female mammals.

[0125] Another aspect described herein is a non-therapeutic method for inducing follicular waves in a female mammal, the method comprising: administering an effective amount of a GnRH antagonist to the female mammal. Another aspect includes a non-therapeutic method for inducing ovulation in a female mammal, the method comprising: I) a) administering an effective amount of a GnRH antagonist to the female mammal; and b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; or II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of a progesterone or a progesterone analog to the female mammal, b) ceasing administration of the progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist, and c) administering an effective amount of an ovulation inducing agent at a fixed time after ceasing administration of the progesterone or a progesterone analog and / or administering the PGF or a PGF analog.

[0126] Another aspect of the present disclosure includes a non-therapeutic fixed-time artificial insemination method, which includes: I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of the GnRH antagonist; and c) artificially inseminating the female mammal at a fixed time after the administration of the ovulation inducing agent; or II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) stopping the administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after stopping the administration of progesterone or a progesterone analog and / or administering PGF or a PGF analog; and d) artificially inseminating the female mammal at a fixed time after the administration of the ovulation inducing agent.

[0127] Another aspect of the present disclosure includes a breeding management method comprising: I) a) administering an effective amount of a GnRH antagonist to a female mammal; b) administering an effective amount of an ovulation inducing agent at a fixed time after administering the GnRH antagonist; and c) mating the female mammal after administering the ovulation inducing agent; or II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of a progesterone or progesterone analog to a female mammal; b) ceasing administration of the progesterone or progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after administering the GnRH antagonist; c) administering an effective amount of an ovulation inducing agent at a fixed time after ceasing administration of the progesterone or progesterone analog and / or administering the PGF or PGF analog; and d) mating the female mammal after administering the ovulation inducing agent. In each of these aspects, the cycle phase of the female mammal need not be known, and / or the method can be used with a mixed population of female mammals.

[0128] Another aspect of the present disclosure includes a gonadotropin stimulation and superovulation method, optionally for oocyte collection or embryo production / transfer, comprising: I) a) administering an effective amount of a GnRH antagonist to a female mammal; and b) administering an effective amount of FSH at a fixed time after the administration of the GnRH antagonist; or) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) administering an effective amount of FSH at a fixed time after the administration of the GnRH antagonist; and c) ceasing administration of progesterone or a progesterone analog and administering an effective amount of a prostaglandin (PGF) or a PGF analog at a fixed time after the administration of FSH. In one embodiment, the method further comprises I) c) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of FSH; or II) d) administering an effective amount of an ovulation inducing agent at a fixed time after the cessation of progesterone or a progesterone analog and / or the administration of PGF or a PGF analog. In one embodiment, multiple oocytes are collected after administration of a GnRH antagonist, FSH, PGF or PGF analogs or an ovulation induction agent, for example, for in vitro fertilization / embryo production. In one embodiment, artificial insemination of an animal is performed after administration of a GnRH antagonist, FSH, PGF or PGF analogs or an ovulation induction agent, and multiple embryos are collected, optionally about 5 to 8 days after artificial insemination. In each of these various aspects and embodiments, the cycle stage of the female mammal need not be known, and / or the method can be used for a mixed population of female mammals.

[0129] Also provided herein is the use of a GnRH antagonist for synchronizing the appearance of follicular waves and / or ovulation in a population of female mammals. Further provided herein is the use of a GnRH antagonist for inducing the appearance of follicular waves in female mammals. Further provided herein is the use of a GnRH antagonist for inducing ovulation in female mammals. Further provided herein is the use of a GnRH antagonist in a fixed-time artificial insemination method. Further provided herein is the use of a GnRH antagonist in a gonadotropin stimulation and superovulation treatment method (optionally used in an embryo production / transfer method). In these aspects, it is not necessary to know the cycle stage of the female mammal, and / or it can be used in a mixed population of female mammals.

[0130] Also disclosed herein is the use of a GnRH antagonist (optionally Cetrorelix) in the manufacture of a medicament for synchronizing the appearance of follicular waves in a population of female mammals. One aspect includes the use of a GnRH antagonist (optionally Cetrorelix) in the manufacture of a medicament for synchronizing ovulation in a population of female mammals. One aspect includes the use of a GnRH antagonist in the manufacture of a medicament for inducing the appearance of follicular waves in female mammals. One aspect includes the use of a GnRH antagonist in the manufacture of a medicament for inducing ovulation in female mammals. One aspect includes the use of a GnRH antagonist in the manufacture of a medicament for fixed-time artificial insemination methods and / or for gonadotropin stimulation and superovulation treatment for embryo production / transfer. In these aspects, the cycle stage of the female mammal does not have to be known, and / or the medicament can be used in a mixed population of female mammals.

[0131] In yet another aspect of the disclosure, a GnRH antagonist (optionally Cetrorelix) is used to synchronize the appearance of follicular waves in a population of female mammals. In one aspect, a GnRH antagonist is used to synchronize ovulation in a population of female mammals. In one aspect, a GnRH antagonist is used to trigger the appearance of follicular waves in female mammals. In one aspect, a GnRH antagonist is used to induce ovulation in female mammals. In one aspect, a GnRH antagonist is used to induce ovulation in female mammals. In one aspect, a GnRH antagonist is used in a fixed-time artificial insemination method. In another aspect, a GnRH antagonist is used in gonadotropin stimulation and superovulation treatment for embryo production / transfer. In these aspects, the cycle stage of the female mammal does not have to be known, and / or the GnRH antagonist can be used in a mixed population of female mammals.

[0132] The term "GnRH antagonist" as used herein refers to an agent that reduces, decreases or otherwise blocks the activity of GnRH and / or GnRH receptors, and includes, but is not limited to, small molecules, peptides and antibodies (and fragments thereof). GnRH antagonists are also referred to as luteinizing hormone-releasing hormone (LHRH) antagonists in the literature. Commonly used GnRH antagonists include, but are not limited to, the following peptides: cetrorelix, abarelix, degarelix, ganirelix, anrelix / teverelix, prarelix, ramorelix, antitide, detirelix, ozarelix, acyline, and the following small molecule compounds: elagolix, linzagolix, relugolix, opigolix, shufgolix and A-75998 (see also Table 24). Thus, in one embodiment, the GnRH antagonist is cetrorelix, abarelix, degarelix, ganirelix, anrelix / teverelix, prarelix, ramorelix, antitide, detirelix, ozarelix, acyline, elagolix, linzagolix, relugolix, opigolix, sufgolix, or A-75998. In one embodiment, the GnRH antagonist comprises abarelix. In one embodiment, the GnRH antagonist comprises degarelix. In one embodiment, the GnRH antagonist comprises relugolix. In one embodiment, the GnRH antagonist is cetrorelix.

[0133] As used herein, "Cetrolex" (trade name: Cetrorelix TM ) refers to the synthetic decapeptide acetyl-D-3-(2′-naphthyl)-alanine-D-4-chlorophenylalanine-D-3-(3′-pyridyl)-alanine-L-serine-L-tyrosine-D-citrulline-L-leucine-L-arginine-L-proline-D-alanine-amide (SEQ ID NO: 2); IUPAC name: N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-L-tyrosyl-D-citrullyl-L-leucyl-L-arginyl-L-prolyl-D-alaninamide.

[0134] As used herein, "Abarelix" (Plenaxis) TM ) refers to N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-N-methyl-L-tyrosyl-D-asparaginyl-L-leucyl-N6-isopropyl-L-lysyl-L-prolyl-D-alaninamide.

[0135] As used herein, "Degarelix" (trade name: Firmagon) TM) refers to N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-4-((S)-dihydroorotamido)-L-phenylalanyl-4-ureido-D-phenylalanyl-L-leucyl-N6-isopropyl-L-lysyl-L-prolyl-D-alaninamide.

[0136] As used herein, "Relugoli" (trade name: Orgovyx TM 、Relumina TM ) refers to 1-[4-[1-[(2,6-difluorophenyl)methyl]-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea.

[0137] The term "follicular wave" refers to a phase in the ovarian cycle that corresponds to the simultaneous growth of multiple follicles, followed by the selection and continued growth of one or more dominant follicles (depending on the species) and the regression of the remaining subordinate follicles. Thus, the terms "follicular wave emergence", "FWE", "wave emergence" and their variants refer to the beginning or early stages of a follicular wave. In cattle and alpacas, FWE can be retrospectively defined as the first detection of dominant follicles of 4 or 5 mm in size, accompanied by an increase in the number of 3-4 mm follicles.

[0138] The phrases "synchronized follicular wave occurrence," "synchronized follicular wave occurrence," and variations thereof, when used with respect to a population of female mammals at different (i.e., random) stages of the ovarian cycle, mean that the follicular wave occurrence in an individual animal occurs at approximately the same time or within the same time span as the follicular wave occurrence in the remaining animals in the population. For example, FWE may occur in an individual animal on the same day or within one or two days as other animals in the population. As will be appreciated by those skilled in the art, synchronized FWE in a population of animals allows ovulation induction methods to be synchronized in the population, thereby providing synchronized ovulation in the population of animals.

[0139] The time of FWE following administration of a GnRH antagonist will vary depending on various factors, such as the given drug or compound, the dose administered, the drug formulation, the route of administration, the species of mammal being treated, etc. The time of occurrence of FWE for a given combination of factors (e.g., species, drug, dose, etc.) can be determined experimentally using methods known in the art, such as the use of ultrasound (e.g., repeated ultrasound examinations at 12 to 24 hour intervals).

[0140] As used herein, the term "fixed time" means a specific or predetermined amount of time between one event (such as a stage or step of a method) and another event (such as another stage or another step in the method). As will be appreciated by those skilled in the art, the time of occurrence of subsequent events or steps in the methods described herein ("fixed time") will depend on various factors (e.g., species, drug, dosage, etc.) as indicated above. For example, a new FWE occurs at a fixed time after administration of a GnRH antagonist. In the examples shown herein, in heifers, new FWE was observed at a fixed time of about 3.40 ± 0.75 days after administration of a single intramuscular dose of cetrorelix acetate at 20 μg / kg body weight, about 3.5 ± 0.4 days after administration of a single intramuscular dose of cetrorelix at 3 mg (about 7.5 μg / kg body weight), or about 5.3 ± 0.3 days after administration of the first dose of cetrorelix at two 1.5 mg (about 3.75 μg / kg body weight) intramuscular doses. In the examples presented herein, in alpacas, new FWE was observed at a fixed time of approximately 7.92 ± 0.24 days after administration of the first of two 1.5 mg intramuscular doses of cetrorelix acetate or approximately 5.33 ± 0.70 days after a single intramuscular injection of 1.5 mg cetrorelix.

[0141] For the methods described herein involving superstimulation or superovulation methods, "administering an effective amount of FSH or FSH agonist to a female mammal at a fixed time after administration of a GnRH antagonist" means that FSH or FSH analogs are administered starting at the expected time of about FWE (produced by administration of a GnRH antagonist) and then continuing for 3 to 7 days depending on the mammalian species. In one embodiment, FSH can be administered by intramuscular injection at intervals of 12 hr for 4 or 7 days starting at the time of FWE. As will be understood, FSH can block the degeneration of existing dominant follicles. Therefore, in order to induce the appearance of a new wave and / or synchronize the appearance of a follicular wave to induce superstimulation or superovulation, FSH should not be co-administered with a GnRH antagonist. In the methods described herein, FSH should be administered only after endogenous LH and / or FSH levels have decreased to baseline levels, and / or after any existing dominant follicles are expected to have degenerated and a new follicular wave has been induced after administration of a GnRH antagonist.

[0142] As will be understood in the art, in some cases, such as in species of spontaneously ovulating animals (e.g., bovine, ovine, equine, porcine, canine), individual mammals in the herd may have existing corpora lutea that may interfere with synchronization. Therefore, to avoid the need to examine individual animals for the presence of existing functional corpora lutea, additional treatments may be administered. For example, progesterone may be co-administered with a GnRH antagonist a) to prevent LH surge and ovulation, b) to prepare the endometrium to prevent early PGF release after ovulation, and c) progesterone withdrawal to establish the ovulatory cascade. Similarly, PGF may be administered at about the time of progesterone withdrawal to induce luteolysis of any existing corpora lutea, which further results in a decrease in plasma progesterone levels and, if a growing or early quiescent dominant follicle is present at the time, triggers the ovulatory cascade. Thus, for the methods described herein, "administering a prostaglandin (PGF) or a prostaglandin analog at a fixed time after administration of a GnRH antagonist" or "administering a prostaglandin (PGF) or a prostaglandin analog at a fixed time after administration of FSH" means administering PGF or a PGF analog at approximately the time when a functional corpus luteum is sensitive to PGF-induced luteolysis. Similarly, administration of progesterone or a progesterone analog will be stopped at approximately the time required to initiate the ovulatory cascade (e.g., when a growing or early quiescent dominant follicle is expected to be present after FWE produced by administration of a GnRH antagonist). For example, in the case of heifers administered a single intramuscular dose of cetrorelix acetate at 20 μg / kg body weight, PGF or a PGF analog is administered at a fixed time of approximately 7-12 days or optionally approximately 7-8 days after administration of the GnRH antagonist (or approximately 3-7 days or optionally approximately 3-4 days after FWE). Similarly, the progesterone or progesterone analog is discontinued about 7-12 days or optionally about 7-8 days after administration of the GnRH antagonist in cattle (or about 3-7 days or optionally about 3-4 days after FWE). Optionally, the progesterone or progesterone analog is discontinued about 0-48 hours after administration of PGF or PGF analog. Those skilled in the art can determine the appropriate time to administer PGF or PGF analog and / or discontinue progesterone or progesterone analog.

[0143] As will be understood in the art, it is preferred to administer an ovulation inducing agent when there is a preovulatory dominant follicle. Therefore, for the methods described herein, "administering an effective amount of an ovulation inducing agent at a fixed time after administering a GnRH antagonist" or "administering an effective amount of an ovulation inducing agent at a fixed time after administering FSH" means administering an ovulation inducing agent approximately at the time when the preovulatory dominant follicle has developed after administering the FWE produced by the administration of the GnRH antagonist. For example, in cattle, the preovulatory dominant follicle is expected to develop about 9-14 days after administering the GnRH antagonist (or about 5-7 days after the expected FWE) or about 5-9 days after administering FSH. Therefore, the ovulation inducing agent can be administered about 9-14 days after administering the GnRH antagonist (or about 5-7 days after the expected FWE) or about 5-9 days after administering FSH. For the methods described herein, "administering an effective amount of an ovulation inducing agent at a fixed time after administration of PGF or a PGF analog," "administering an effective amount of an ovulation inducing agent at a fixed time after discontinuation of progesterone or a progesterone analog," or "administering an effective amount of an ovulation inducing agent at a fixed time after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog" means administering an ovulation inducing agent after luteolysis and / or preovulation / LH surge of any pre-existing corpus luteum after administration of PGF or a PGF analog and / or discontinuation of progesterone or a progesterone analog. For example, in the case of heifers, luteolysis and / or preovulation / LH surge of the corpus luteum occurs approximately two days after administration of PGF or a PGF analog and / or discontinuation of progesterone or a progesterone analog. Thus, the ovulation inducing agent may be administered approximately 2 days after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog. The appropriate time to administer the ovulation inducing agent may be determined by a skilled person.

[0144] For the methods described herein involving breeding / mating or artificial insemination, "at a fixed time after the administration of an ovulation induction agent" means mating or artificial insemination within a period of time relative to the expected ovulation produced by the administration of the ovulation induction agent, such as before the expected ovulation, about the expected ovulation time and / or within a period of time after the expected ovulation. The time when ovulation occurs may depend on the ovulation induction agent, the dosage and the animal species, and the time when mating or artificial insemination occurs (before the ovulation time, about the ovulation time and / or after the ovulation time) depends on the species. For example, in alpacas, ovulation occurs about 24 to 30 hr after the administration of LH (regardless of mating or not), and mating or artificial insemination of alpacas is performed about 12-24 hr before ovulation. Therefore, mating or artificial insemination of alpacas can be performed 0 to 18 hours after the administration of LH, such as about 12 hours after the administration of LH. Mating or artificial insemination can be performed, for example, in cattle, sheep, goats and alpacas, about 12-24 hr before ovulation, or, for example, in dogs, about 24-36 hr after ovulation. In cattle, mating or artificial insemination is performed up to about one day after administration of an ovulation induction agent, for example, at about the same time as administration of an ovulation induction agent (e.g., GnRH or LH), about 12 hours and / or about 24 hours after administration of an ovulation induction agent. As will be appreciated, the appropriate time for mating or artificial insemination will depend on the species of the mammal and can still be determined by a skilled person.

[0145] For the methods described herein involving oocyte collection, "collecting one or more oocytes at a fixed time after administration of a GnRH antagonist", or "collecting one or more oocytes at a fixed time after administration of FSH or an FSH agonist", or "collecting one or more oocytes at a fixed time after administration of an ovulation inducing agent" means collecting oocytes when the expected oocytes are at the desired developmental stage. For example, if FSH is not used, immature oocytes can be collected about 2-4 days after the expected FWE, or if FSH is administered, immature oocytes can be collected about 3-9 days after FWE. Any suitable method can be used to collect oocytes, such as by using a vacuum to aspirate oocytes (including cumulus-oocyte complexes) and follicular fluid from the ovaries by ultrasound-guided puncture with a needle. For example, in cattle, oocytes may be collected 6-11 days after GnRH antagonist treatment without FSH treatment, or 3-9 days after the start of FSH treatment, or 30-48 hours after the withdrawal of progesterone or the administration of PGF, or 0-24 hr after the intramuscular injection of an ovulation induction agent such as GnRH or LH. As will be appreciated, the appropriate time for oocyte collection depends on the species of mammal and the desired stage of oocyte development, and remains determined by the skilled artisan.

[0146] For the methods described herein involving embryo collection, "collecting one or more embryos at a fixed time after artificial insemination" or "collecting one or more embryos at a fixed time after mating" means collecting embryos when the expected embryos are at the morula or blastocyst stage. Any suitable method can be used, such as a surgical method, or in a non-surgical manner, by placing a catheter through the vagina and cervix into the uterus or uterine horns, and collecting embryos from the uterus. Embryos can be collected, for example, about 5 to 10 days after artificial insemination. For example, in cattle, embryos are collected 7, 8 and / or 9 days after artificial insemination. As will be appreciated, the appropriate time for embryo collection depends on the species of the mammal and the desired stage of embryonic development, and can still be determined by a technician.

[0147] For the method related to embryo transfer as described herein, "transferring the embryo to a female mammal at a fixed time after administering an ovulation inducing agent" means transferring the embryo after ovulation and subsequent corpus luteum development producing progesterone, which can be, for example, about 2 to 8 days after the expected ovulation time, or about 3 to 10 days after administering an ovulation inducing agent, depending on species. Any suitable embryo can be transferred, including fresh / unfrozen or frozen-thawed embryos collected in vivo or embryos produced in vitro. Embryos can be transferred using any suitable method known in the art, such as by surgical methods, or in a non-surgical manner, by placing a catheter through the vagina and cervix into the uterus or uterine horns, embryos are transferred to the uterus or uterine horns. According to the type and stage of the embryo, embryos can be transferred to a recipient for example about 3 to 10 days after administering an ovulation inducing agent. For example, in cattle, embryos can be transferred 7, 8 or 9 days after administering LH, GnRH or estradiol. As will be understood in the art, progesterone (e.g., produced by corpus luteum) acts on the endometrium that develops synchronously with any developing embryo, so the age of the embryo should be matched with the number of days after ovulation (e.g., within about 24 hours). For example, if about 7 days after ovulation, the embryo is transferred to the receptor, then the embryo should be in about 7 days of development. For example, in people, the zygote can be directly transferred to the uterus 2 days after ovulation, and the blastocyst can be transferred to the uterus 5 to 6 days after ovulation. Therefore, it should be understood that the appropriate time for embryo transfer depends on many factors, including mammalian species, ovulation inducing agents and the stage of the transferred embryo, and still can be determined by technicians.

[0148] As used herein, the term "mammal" is suitably used to refer to animal species in which female animals undergo an ovulation cycle (e.g., estrus). Unless the context clearly specifies otherwise, the term "animal" is also used herein to refer to mammals, and in some contexts is particularly used to refer to female mammals, including humans. The term "domestic animal" refers to mammal species that are usually raised or kept in captivity, such as, for example, livestock / pack animals (e.g., bovines, such as cattle and buffaloes, camelids, such as llamas, alpacas, and camels, sheep (e.g., sheep), capra (e.g., goats), suidae (e.g., pigs), equines (e.g., horses and donkeys)) and pets (e.g., cats, dogs), or particularly female animals of these species. Similarly, the term "wild animal" includes semi-domesticated and non-domesticated mammal species, such as, for example, bison, elk, reindeer, deer (e.g., white-tailed deer), musk oxen, non-human primates, canines, and felines, such as tigers, lions, black panthers, or female animals of these species. In one embodiment, the mammal is a domesticated animal, optionally a bovine, camelid, ovine, caprine, porcine, canine, or feline. In one embodiment, the mammal is a wild animal, optionally a bison, elk, reindeer, deer, musk ox, non-human primate, canine, or feline. In one embodiment, the mammal is a human.

[0149] As used herein, the term "mixed population" means a population of female mammals that are asynchronous in their ovulatory cycle phases.

[0150] As used herein, the term "oocyte" means a female reproductive cell of any stage that can produce an embryo once fertilized, and includes, but is not limited to, oocytes, eggs, and cumulus-oocyte complexes (e.g., an oocyte surrounded by a dense or expanded granulosa / cumulus cell layer).

[0151] As used herein, the term "embryo" means an animal in an early stage of development, such as a zygote, morula, or blastocyst.

[0152] The term "administered or administering" as used herein means administering an effective amount of a compound or composition of the present disclosure to an animal. The term "co-administration" or "combination therapy" shall mean administering at least two compounds or compositions to an animal so that each of the two or more compounds can be found in an effective amount or concentration in the animal at a given time point. Although the compounds according to the present disclosure can be co-administered to an animal simultaneously, the term includes administering two or more agents simultaneously or at different times, provided that all co-administered compounds or compositions are found in effective concentrations in the animal at a given time.

[0153] As used herein, the phrase "effective amount" means an amount effective to achieve the desired result at the necessary dosage and time period. For example, in the case of follicular wave emergence (FWE), an effective amount is an amount that induces FWE in a female mammal at an approximately fixed time (or within a consistent time range, such as within (±) 1 or 2 days) after administration compared to the response obtained without administration of the compound. The effective amount may vary depending on factors such as the species, age, reproductive status and / or body weight of the animal. The amount of a given compound corresponding to such an amount will vary depending on various factors, such as a given drug or compound, drug formulation, route of administration, species of mammal being treated, etc.

[0154] The GnRH antagonist can be administered according to any suitable schedule, for example, as a single dose or multiple doses (e.g., two doses), separated by a suitable period of time (e.g., about one day), or any other suitable number of doses and / or time periods, depending, for example, on a given drug or compound, formulation, route of administration, and / or species of mammal. In one embodiment, the GnRH antagonist is administered as a single dose. In one embodiment, the GnRH antagonist is administered in two doses separated by about one day. In one embodiment, the GnRH antagonist is administered as a single dose in a dosage form that provides extended or continuous release over a period of time (e.g., over a period of about one day, about two days, about three days, or longer).

[0155] The GnRH antagonist can be administered by any suitable route of administration and using any suitable dosage form, for example, by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, administration by inhalation or spray (e.g., via an aerosol), mucosal administration, rectal administration, vaginal administration, skin patch or dermal application, or oral administration, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0156] Compositions comprising GnRH antagonists such as Cetrorelix can be provided in any suitable dosage form. The term "dosage form" as used herein refers to, for example, the physical form of a dose comprising a compound of the present disclosure, and includes, but is not limited to, injectable dosage forms, including, for example, sterile solutions suitable for preparation for injection and sterile powders for reconstruction, etc.; and liquid and solid dosage forms, including, for example, tablets, caplets, soft capsules, capsules, ingestible tablets, buccal tablets, lozenges, elixirs, suspensions, syrups, wafers, resuspendable powders, skin patches, intravaginal or subcutaneous implants, impregnated silicone devices, liquids and solutions. For example, an injectable dosage form can be a subcutaneous, intradermal or intramuscular depot injection, which allows the compound to be released in a controlled and consistent manner over a period of time (e.g., over a few days or weeks). Methods for preparing depot injections are described, for example, in U.S. Pat. No. 3,089,815, entitled "Injectable pharmaceutical preparation, and a method of making the same," and incorporated herein by reference in its entirety. Dosage forms may also include those suitable for mucosal administration, including, for example, gels, creams, ointments, foams, tablets or capsules, or via inserts comprising, for example, a substrate coated or impregnated with a GnRH antagonist (e.g., a silicone coating, a plug or a sponge), suitable for vaginal administration, or skin patches.

[0157] When administered by intramuscular injection, examples of suitable dosage ranges for cetrorelix may include, for example, about 2 μg / kg body weight to about 200 μg / kg body weight, about 5 μg / kg body weight to about 100 μg / kg body weight, about 7.5 μg / kg body weight to about 75 μg / kg body weight, about 20 μg / kg body weight to about 50 μg / kg body weight, or any suitable dosage or dosage range between about 2 μg / kg body weight to about 200 μg / kg body weight, such as about 2.5 μg / kg body weight, about 3 μg / kg body weight, about 4 μg / kg body weight, about 5 μg / kg body weight, about 7.5 μg / kg body weight, about 10 μg / kg body weight, about 15 μg / kg body weight, about 20 μg / kg body weight, about 25 μg / kg body weight, about 30 μg / kg body weight, about 35 μg / kg body weight, or about 40 μg / kg body weight. In one embodiment, the effective dose of Cetrorelix administered by intramuscular injection is about 7.5 μg / kg body weight. In one embodiment, the effective dose administered by intramuscular injection is about 40 μg / kg body weight. The dosage can be administered as a single dose or in multiple doses separated by a period of time (such as, for example, a 24-hour period). In one embodiment, Cetrorelix of about 7.5 μg / kg body weight is administered as a single intramuscular dose. In one embodiment, Cetrorelix of about 7.5 μg / kg body weight is administered as two intramuscular doses of about 3.75 μg / kg body weight given at intervals of about 24 hours. In one embodiment, Cetrorelix of about 20 μg / kg body weight is administered as a single intramuscular dose. In one embodiment, Cetrorelix of about 40 μg / kg body weight is administered as two intramuscular doses of about 20 μg / kg body weight given at intervals of about 24 hours.

[0158] GnRH antagonists can be used or used in combination with one or more additional drugs to provide enhanced or additional reproductive management control, such as for inducing ovulation at a fixed time after the administration of the GnRH antagonist (which can be synchronized in a mammalian population to synchronize ovulation). Controlled or synchronized ovulation can be applied to breeding management, fixed-time artificial insemination schemes or embryo transfer schemes. For example, GnRH antagonists can be optionally used in combination with progesterone or progesterone analogs, while subsequently administering prostaglandins (PGF) or prostaglandin analogs, and / or subsequently administering ovulation induction agents to delay and / or enhance ovulation synchronization after the follicular wave induced by the GnRH antagonist. Such combinations may be particularly suitable for fixed-time artificial insemination or embryo transfer schemes. Therefore, in one embodiment, the method further includes administering an effective amount of one or more additional drugs. In one embodiment, one or more additional drugs include progesterone or progesterone analogs, prostaglandins (PGF) or PGF analogs and / or ovulation induction agents. Other drugs that can be used in addition to GnRH antagonists in the methods described herein include, but are not limited to, LH, GnRH, PGF2a, FSH, equine chorionic gonadotropin (eCG), human chorionic gonadotropin (hCG), and estradiol benzoate, depending on the intended use. For example, fixed-time AI and embryo transfer differ in the use of FSH, PFG2a, and the dosage of LH / GnRH. For oocyte collection, LH can be omitted.

[0159] Progesterone and progesterone analogs can be administered using progesterone drug release devices, including commercial products such as controlled internal drug release (CIDR) devices (Zoetis), PRID TM (CEVA), cue-mate TM (Vetoquinol) and subcutaneous devices such as syncromate-B. Progesterone analogs such as Norgestomet or oral supplementation of MGA (Melengestrol Acetate) can also be used in combination with GnRH antagonists.

[0160] Prostaglandins (PGF) and PGF analogs include, but are not limited to, prostaglandin 2 alpha (PGF2a), cloprostenol, dinoprost, dinoprost tromethamine, bimatoprost, travoprost, carboprost, and latanoprost.

[0161] As used herein, "ovulation induction agent" means an agent that, when administered to a female mammal, causes ovulation of any existing preovulatory dominant follicle present in the female mammal. Ovulation induction agents include, but are not limited to, GnRH and GnRH analogs and agonists (e.g., buserelin, deslorelin, futirelin, gonadorelin, goserelin, leuprorelin, triptorelin), LH and LH analogs, agonists, conjugates and recombinant products (e.g., human chorionic gonadotropin / hCG, equine chorionic gonadotropin / ECG), as well as estradiol and estradiol esters, analogs and agonists (e.g., estradiol-17β, estradiol benzoate, estradiol valerate, estradiol cypionate). Suitable ovulation induction agents can be selected by a technician and can depend on the species of the mammal. For example, ovulation induction agents suitable for camelids include GnRH or LH, but do not include estradiol.

[0162] III. Devices and Kits

[0163] One aspect includes a drug release device comprising a GnRH antagonist. Suitably, the drug release device is configured to provide a controlled release of an effective amount of a GnRH antagonist over a period of time (such as, for example, about one day to about three days). In one embodiment, the GnRH antagonist is cetrorelix. In one embodiment, the drug release device further comprises a progesterone, and is optionally configured to provide a controlled release of an effective amount of a progesterone over an extended period of time (such as, for example, at least about 5 days to at least about 10 days or longer, optionally, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or longer).

[0164] One aspect includes a test kit comprising a GnRH antagonist and a suitable container or packaging and / or instructions for use thereof, such as, for example, for synchronizing FWE and optional ovulation in a female mammal population, for a fixed time protocol, such as a fixed time artificial insemination protocol, a fixed time oocyte collection protocol, or a fixed time embryo production or embryo transfer protocol, or for gonadotropin stimulation and superovulation treatment protocols, optionally for embryo production / transfer. In one embodiment, the GnRH antagonist is provided in a drug release device. In one embodiment, the test kit further comprises an applicator or other suitable delivery device. In one embodiment, the test kit further comprises one or more additional drugs, such as, for example, progesterone, PGF2A or its analogs, such as cloprostenol, GnRH, eCG and / or LH.

[0165] III. Embodiment

[0166] The following non-limiting examples illustrate the present application:

[0167] Example 1. Effect of GnRH antagonist (Cetrolix) on the response to sutrin treatment

[0168] Kissin is the neuropeptide product of the Kiss-1 gene, which is cleaved and / or degraded into 54-amino acid peptides, 14-amino acid peptides, 13-amino acid peptides, and 10-amino acid peptides (Kotani et al. 2001). GnRH and kissin immunoreactive cells are closely associated in the preoptic area and arcuate nucleus of the hypothalamus of mice, primates, sheep (Ramaswamy et al. 2011; Smith et al. 2008; Clarkson and Herbison, 2006) and cattle (Tanco et al. 2016). Peripheral injection of kisspeptin induces luteinizing hormone (LH) secretion and ovulation in several mammalian species (Caraty et al. 2007; Caraty et al. 2013; d'Anglemont de Tassigny et al. 2008; Ezzat Ahmed et al. 2009; Matsui et al. 2004), including cows (Leonardi et al. 2019; Leonardi et al. 2018) and seasonally estrous sheep (Caraty et al. 2007). In addition, intravenous administration of kisspeptin-10 induces GnRH release into the hypophyseal portal circulation of sheep (Smith et al. 2011). It is unclear whether the 10-amino acid kisspeptin fragment (kissin-10) can cross the blood-brain barrier to stimulate GnRH neurons after peripheral injection in cattle.

[0169] It is not known whether peripheral kisspeptin can activate bovine GnRH neurons.

[0170] Endogenous and exogenous GnRH induces the release and synthesis of LH from the bovine pituitary gland (Vizcarra et al. 1997; Yoshioka et al. 2001), and administration of GnRH antagonists prevents the LH surge (Ginther et al. 2012). Peripheral administration of kissin increases plasma LH concentrations in ovariectomized cows (Whitlock et al. 2008) and pubertal heifers (Kadokawa et al. 2008) and induces ovulation in a low progesterone environment (Leonardi et al. 2019). In a previous study, a progressive increase in LH secretion was detected within 15 minutes after repeated intravenous administration of kissin-10 in cows with plasma progesterone below 1.7 ng / ml (Leonardi et al. 2019). However, the mechanism by which peripheral administration of kissin-10 induces LH release in cows is unclear. In vitro studies in horses and cattle raise the possibility of direct release of LH from anterior pituitary cells by kissin (Ezzat et al. 2010; Magee et al. 2020). It remains unknown whether kisspin-10 crosses the blood-brain barrier to stimulate GnRH neuronal cell bodies or acts on GnRH nerve terminals in the median eminence, or whether the observed LH release is due to a direct effect on pituitary gonadotrophs in vivo.

[0171] Objective: To determine whether pretreatment with a GnRH antagonist alters the pattern of kisspeptin-induced LH release and ovulation. Cetrorelix was used as a blocker of kisspeptin action in a model of persistent dominant follicles with low plasma progesterone.

[0172] Hypothesis: 1) Pretreatment with a GnRH antagonist (cetrorelix) prior to kisspeptin treatment will inhibit LH release and prevent ovulation in cows.

[0173] Methods: Human kissin-10 (hKP-10) peptide (YNWNSFGLRF-NH2; SEQ ID NO: 1) was custom synthesized by GenScript USA Inc, Piscataway, NJ, USA at >95% purity (MW: 1318.44 g / mol). The sequence is based on the predicted C-terminal region (112-121-NH2) of human metastin (GenBank Accession No. AY117143) and has been previously used in cattle. The solubility of the peptide was pre-tested and dissolved in ultrapure water at 10 mg / mL.

[0174] Hereford crossbred heifers (n=15; 500±24 kg body weight, 17-18 months of age) were studied, with a corpus luteum in one ovary at the start of the experiment. The heifers were maintained in outdoor pens at the University of Saskatchewan Goodale Research Farm (52°N and 106°W). The heifers were fed barley silage and had ad libitum access to hay and water. Mineral salt blocks were available to the heifers throughout the study. All procedures were performed in accordance with the Canadian Council on Animal Care and approved by the University of Saskatchewan Protocol Review Committee.

[0175] A schematic diagram of the experimental design is shown in Figure 1In. Heifers (n=15) were selected from a larger group based on the detection of corpora lutea (CL) by transrectal ultrasonography. Immediately after ultrasonography, heifers were given PGF2α twice at 12-hour intervals. The hypothesis was tested using a previously described low progesterone bovine model (Leonardi et al., 2019). Briefly, ovaries were examined daily by ultrasonography to detect ovulation. Three days after ovulation, follicles with a diameter of ≥5 mm in both ovaries were ablated by transvaginal ultrasound-guided follicular aspiration, inducing the emergence of new follicular waves 1.5 days after the procedure (Bergfelt et al. 1994). After follicular ablation, a progesterone device was placed in the vagina (CIDR; Zoetis). The day of wave emergence (day 0) was defined as the day when the follicular wave (4 to 5 mm in diameter) was first recorded with an increase in diameter. Heifers were given PGF2α im on days 3.5 and 4 (i.e., 8 and 8.5 days after ovulation) to induce luteolysis, however, the CIDR device was left in place until day 12 to maintain a low progesterone environment sufficient to prevent spontaneous ovulation. On day 6, an indwelling jugular catheter was placed as described (Bergfelt et al. 1997), and heifers were randomly divided into three groups (n=5 per group): 1) 3 iv doses of 15 mg human kissin-10 at 1 hour intervals (Kp10 group), 2) pre-treated with a single im dose of cetrorelix acetate (20 μg / Kg body weight, GnRH antagonist-Sigma No. C5249 diluted in 5% D-mannitol w / v (Sigma No. M4125)) followed by treatment 3 hours later with 3 iv doses of 15 mg human kissin-10 at 1 hour intervals (cetrorelix group), or 3) 3 iv doses of saline at 1 hour intervals (control group). After treatment (on day 6), ovarian ultrasound examinations were performed daily until the end of the experiment to detect ovulation of the existing dominant follicle (i.e., the dominant follicle present at the time of treatment) or from the dominant follicle of the subsequent wave. The progesterone device was removed on day 12 (i.e., 13 days after insertion), and ultrasound examinations were continued until ovulation was detected.

[0176] Blood samples and hormone determination: Serial blood samples on the day of treatment were collected at 15-minute intervals using an indwelling jugular vein catheter from -30 to 150 min (0 min = time of first HKP-10 or saline injection). All blood samples were collected in heparinized tubes (Vacutainer, BD, Franklin Lakes NJ, USA). Immediately after sampling, the tubes were centrifuged at 1,500 × g for 15 min, plasma was separated and stored at -20°C.

[0177] Plasma samples from two experiments were analyzed for LH and progesterone concentrations in the research laboratory of Dr. O. J. Ginther at the University of Wisconsin (Madison, WI, USA). Plasma LH concentrations were measured using a radioimmunoassay validated for use in cattle (Bolt et al. 1990) and modified as reported (Ginther et al. 1999). Briefly, the LH concentration was measured using a radioimmunoassay for use in cattle (Bolt et al. 1990). 125 I-iodinated and used to prepare the reference standard USDA-bLH-B-6, and LH concentrations were measured in duplicate using USDA-309-684P as the primary antibody (National Hormone and Pituitary Program, Torrance, CA, USA). The standard curve ranged from 0.078 to 20.0 ng / mL with a sensitivity of 0.1 ng mL -1 The intra- and inter-assay coefficients of variation and mean sensitivity were 6.23%, 12.24%, and 0.03 ng / mL, respectively. The antibody-coated tubes and 125 Progesterone concentrations were measured using a commercial solid phase RIA kit for I-labeled progesterone (ImmuChem Coated Tube Progesterone 125 RIA Kit, MP Biomedical, Costa Mesa, CA). The intra-assay coefficient of variation and sensitivity for progesterone were 11.97% and 0.06 ng / mL, respectively.

[0178] Statistical analysis: Data analysis was performed using SAS (Statistical Analysis System, Software Package 9.4, SAS Institute Inc., Cary, NC, USA). In both experiments, single point measurements (i.e., diameter of dominant follicle at treatment and 24 hours after treatment, diameter of CL at treatment, and progesterone concentration at treatment) were analyzed using one-way ANOVA. Statistical significance was assumed when p values ​​were ≤ 0.05, and a trend toward a difference was assumed when p values ​​were > 0.05 to ≤ 0.1. Multiple comparisons were performed using Tukey post hoc tests if the p values ​​tested detected a difference. Ovulation rates were analyzed using the GLIMMIX program.

[0179] The MIXED model procedure was used for the analysis of repeated measures data (e.g., LH plasma levels, follicle dynamics), where treatment, time, and treatment-time interactions were tested, and a repeat statement (repeat day object = cow ID) was included in the syntax. Five covariance structures (SIMPLE, CS, AR(1), ANTE(1) or UN) were tested in the initial analysis, and the model with the smallest AICC value was selected for the final analysis. All values ​​are reported as mean ± SEM.

[0180] Results: The diameter of the dominant ovarian follicle did not differ between Kp10, cetrorelix, and the control group at the time of treatment (day 6 of the follicular wave) and 24 h after treatment (Table 1). Plasma progesterone concentrations were similar between the groups at the time of treatment. The Kp10 group had higher plasma LH concentrations than cetrorelix and the control group (p < 0.001, Figure 2 B). From 15 to 240 min, the plasma LH concentration of the Kp10 group remained higher than that of the other two groups (0 min = first injection treatment, Figure 2 A). Ovulation of an existing dominant follicle was recorded in 4 of 5 heifers in the Kp10 group compared to 0 of 5 heifers in the Cetrorelix and Control groups (Table 1). New follicular waves appeared immediately in all four heifers that ovulated after Kissin treatment ( Figure 2 A). In the cetrorelix group, regression of the existing dominant follicles was recorded 3 to 4 days after treatment ( Figure 2 A). The appearance of a new follicular wave (i.e., no ovulation of an existing dominant follicle) occurred at 3.40 ± 0.75 days after treatment in the Cetrorelix group and at 2.75 ± 0.75 days in the Kp10 group (after ovulation of an existing dominant follicle in 4 of 5 cows). In the control group, the existing dominant follicle did not degenerate and ovulated on day 15 after CIDR removal (removal on day 12). All new dominant follicles from the Kp10 (n=4) and Cetrorelix (n=5) groups ovulated on day 15 after CIDR removal (on day 12).

[0181] Table 1: Ovarian and endocrine responses of heifers treated intravenously with human Kp10 (Kp10 group), pretreated with cetrorelix before Kp10 (cetrorelix group), or treated with saline (control group) at subluteal levels of plasma progesterone (mean ± SEM).

[0182]

[0183] a,b Within a row, values ​​without a common superscript differ (P ≤ 0.05)

[0184] *Statistical analysis of two treatment groups

[0185]

[0186] # Dominant follicles present during treatment

[0187] A new wave of dominant follicles appears after treatment

[0188] In 5 of 5 heifers in the Cetrorelix + KP10 group, a new wave occurred during the mid-quiescent dominance period in the low progesterone environment at 3.40 ± 0.75 days post-treatment.

[0189] The wave appearance is attributed to the regression of the existing dominant follicle caused by low plasma LH levels. The new wave of dominant follicles is able to ovulate after CIDR removal.

[0190] In all saline treated control heifers (n=5), dominance of existing follicles was maintained and no new waves occurred. When the CIDR device was removed on day 12, the existing dominant follicles were able to ovulate.

[0191] In 4 of 5 heifers in the KP10 group, a new wave occurred 2.75 ± 0.75 days after treatment due to ovulation of the existing dominant follicle (produced by elevated plasma LH levels). The CL resulting from ovulation of the existing dominant follicle was transient.

[0192] Conclusions: Unexpected downstream effects of LH suppression on the dominant follicle were also observed in cetrorelix treated heifers - all heifers in this group failed to ovulate (0 of 5) compared to 4 of 5 heifers that ovulated 48 hours after kissin treatment. Interestingly, the dominant follicle in all animals entered the catagen phase 72 hours after kissin and GnRH antagonist treatment.

[0193] In conclusion, pretreatment with the GnRH antagonist cetrorelix 3 h before starting intravenous treatment with kissin inhibited the kissin-induced LH surge, prevented ovulation, and caused regression of the existing dominant follicle, followed by the emergence of a new follicular wave on average 3.4 days after treatment in all five cows. A possible mechanism of kissin-mediated LH release is downstream GnRH synthesis, possibly by inducing release of presynthesized GnRH from nerve terminals in the median eminence.

[0194] Example 2. Alpaca Cetrorelix Known Phase Study

[0195] Cetrorelix inhibits the preovulatory LH surge induced by ovulation inhibitory factor (OIF) in llamas, suggesting that LH secretion is regulated by direct or indirect effects of OIF on GnRH neurons in the hypothalamus (Silva et al. 2011). It is unclear whether the effects observed on the dominant follicle in Example 1 also apply to llamas. Furthermore, it is unclear whether the same effects can be observed at other stages of follicular development (growing, quiescent, regressing preovulatory) and other stages of corpus luteum development (metestrus, diestrus, and proestrus).

[0196] Objective: To identify the growth pattern of dominant follicles in camelids after treatment with a GnRH antagonist during different phases of the follicular wave.

[0197] It was hypothesized that: 1) GnRH antagonists would cause regression of both early / mid-anagen and mid-quiescent dominant follicles; and 2) new waves would appear at a consistent time after treatment, regardless of the follicular status of the dominant follicle.

[0198] Methods: The specific objective of this experiment was to identify the growth pattern of dominant follicles in alpacas following treatment with a GnRH antagonist during two contrasting phases of the follicular wave. Based on data from natural spontaneous waves in alpacas, the dominant follicle was expected to be in early to mid-growth phase (6 to 7 mm in diameter) on day 5 and in mid-quiescence phase (9 to 10 mm) on day 10, so treatment was planned to begin on these days.

[0199] The experimental design is shown in Figure 3 In. Transrectal ovarian ultrasonography was performed using an Esoate My Lab 5 ultrasound machine and a 7.5 linear array transducer attached to a custom-built rigid PVC tube handle. The tube was slightly bent at the end of the transducer to facilitate manipulation of the transducer. The transducer and plastic handle were lubricated with methylcellulose ultrasound gel and slowly inserted into the rectum after manually removing feces. The reproductive tract and ovaries were located, and 10 to 20 seconds of video clips of the left and right ovaries were recorded. Ovarian sketches were drawn during imaging every day to record the location, size and number of follicles with a diameter>2mm and the size and location of the corpus luteum (if present). Ultrasound examinations were performed every day. When a new follicle wave (defined as an existing follicle wave) was detected in the animal spontaneously, experimental data were recorded. The day when the wave appeared was designated as day 0, and was retrospectively defined as the first detection of a dominant follicle of 4 or 5mm size, with an increase in the number of 3-4mm follicles.

[0200] Based on the day of the wave, the alpacas were assigned to one of three treatment groups (n=7 animals per group) using a systematic random sampling method. The first group of alpacas (control group) were not treated, their ovaries were examined daily, and blood samples were obtained daily from day 4 to day 12 to compare hormone data from similar days after cetrorelix treatment. The second group of alpacas (D5 group) were given a first intramuscular injection of 1.5 mg (3 mL of 0.5 mg / mL solution) of cetrorelix acetate on day 5, and a second injection of 3 mL was given on day 6 (i.e., two i / m injections of 1.5 mg cetrorelix at 24 hour intervals, for a total dose of 3 mg). The dose of cetrorelix administered was approximately equal to 20 μg / Kg body weight / day (considering the average body weight of alpacas=75 kg) and was based on previous studies in llamas and cattle (Silva et al., 2011, Ulker et al. 2001). Alpacas in the third group (D10 group) were given a first intramuscular injection of 1.5 mg cetrorelix acetate on day 10 and a second injection of 1.5 mg on day 11.

[0201] Cetrorelix treatment was given during the existing follicular wave. Changes in the size of the dominant follicle, the largest subordinate follicle, and the number of follicles ≥3 mm were recorded daily from the spontaneous appearance of the existing follicular wave to day 6 of the next (post-treatment) follicular wave. The interwave interval was determined based on the duration between the appearance of consecutive follicular waves.

[0202] Blood samples (5 to 8 mL) were collected in heparinized tubes (BD Vacuum Blood Collection Tubes, BD Franklin Lakes, NJ, USA) via jugular vein puncture. Blood samples were kept on ice packs until centrifuged at 200 rpm (within 1 hr of collection) to obtain plasma samples, which were immediately frozen at -20 ° C until plasma LH was measured. Plasma samples were obtained on days 4 to 7 (i.e., 1 day before cetrorelix treatment to 1 day after treatment) and on days 9 to 12 in group 2. For direct comparison, plasma samples were obtained from day 4 to day 12 in the control group.

[0203] Results: The results are presented in Table 2. Post-treatment data from one animal in the D10 group were excluded because ovulation occurred on day 10 before the start of treatment.

[0204] In the untreated control group (n=7), the next wave appeared at 14.57±0.81 days after the first wave appeared, i.e., the interval between existing waves ranged from 13 to 19 days, with a median of 14 days. On average, the dominant follicle entered quiescence at 7.6±0.55 days (combined data from the control group and D10 group).

[0205] The average starting day of dominant follicle degeneration in D5 group was earlier than that in D10 group and control group (8.7±0.89 days, 12.9±0.34 days, 13.6±0.90 days, respectively; P<0.001), and the dominant diameter in D5 group was smaller than that in D10 group and control group (7.4±0.5 mm on 6.0±0.90 days, 10.9±0.70 mm on 10.3±0.68 days, 10.2±0.93 mm on 10.0±0.90 days; P=0.005).

[0206] The duration of the dominant follicle quiescence phase was the shortest in the D5 group, intermediate in the D10 group, and longest in the control group (3.9±0.63 days, 4.9±0.40 days, 6.3±0.52 days; P=0.015).

[0207] The interwave interval was shorter in the D5 group, intermediate in the control group, and longer in the D10 group (12.7±0.36 days, 14.57±0.81 days, 17.71±0.52 days, P<0.001).

[0208] In Groups 2 and 3 combined, wave appearance occurred at a mean of 7.92 ± 0.24 days (range 7 to 9 days) after the first cetrorelix injection.

[0209] Taking all the data together, when cetrorelix treatment was given on day 5 of the wave (n=7), the dominant follicle stopped growing, had a shorter resting phase, reached its maximum diameter earlier, and began to degenerate earlier than the control group or the D10 group. A new wave was induced after 7.71±0.36 days (on day 12.7 of the existing wave; i.e., earlier than the control group).

[0210] When cetrorelix treatment was given on day 10 of the wave, the dominant follicle regressed at the same time as the control, and a new wave started 8.17±0.31 days after cetrorelix treatment (on day 18.17 of the existing wave, ie, delayed relative to the control group).

[0211] The synchrony of wave appearance (defined as the interval from the first treatment to the appearance of the next wave) was similar in the D5 and D10 groups (7.7±0.36 days [range: 7-9 days] vs. 7.7±0.52 days [range: 7-9 days, P>0.99]).

[0212] Table 2. Follicular endpoint and wave emergence data (mean ± standard error of the mean) from untreated alpacas (control) and alpacas treated twice with 1.5 mg cetrorelix acetate i / m at 24 hr intervals starting on day 5 (D5) or day 10 (D10) of the follicular wave. The existing follicular wave was defined as the wave during the start of cetrorelix treatment. The reported p values ​​(last column) were obtained by the GLM procedure in SAS. Different superscripts in a row indicate differences between groups detected by Tukey post hoc comparisons (P<0.05).

[0213]

[0214]

[0215] #, n = 6 alpacas

[0216] Conclusions: When treatment was initiated during the growth phase or late quiescent phase of the dominant follicle in alpacas, the dominant follicle regressed and the appearance of the next wave was synchronized after 2 injections of cetrorelix.

[0217] Example 3: Alpaca Random Day Study

[0218] Objective: To examine the effect of a single injection of cetrorelix on the synchronization of dominant follicle regression and next wave emergence in alpacas when treatment was initiated on a random day of the wave.

[0219] Hypothesis: A single treatment with a GnRH antagonist causes regression of dominant follicles at a random developmental stage and re-examine whether new waves appear at a consistent time after treatment regardless of dominant stage (ie, similar to Example 2).

[0220] Methods: Ultrasound examination methods and follicular endpoint recording are similar to those described in Example 2. Follicular endpoints were obtained from alpacas (n=15) before and after treatment, so each animal served as its own control. A fixed calendar day (July 16) was pre-specified for cetrorelix treatment. From May 31 to June 16, daily transrectal ultrasound examinations (n=15 alpacas) were performed. This examination period was used to record changes in wave appearance from a fixed calendar date (May 31) without treatment (control period). Animals were on an unknown / random day of the wave at the beginning of this 17-day time window (i.e., longer than one natural inter-wave interval); therefore, all animals were expected to have at least one wave appearance covering this period. All alpacas were treated with a single intramuscular injection of 1.5 mg (3 mL) of cetrorelix acetate on July 16, and ultrasound examinations continued every day from the day of treatment until the 3rd day (treatment period) after the next (post-treatment) wave appearance. The synchronization of wave appearance was compared between the control period and the treatment period.

[0221] Statistical analysis: Proc Mixed repeated measures analysis of variance (SAS version 9.4) was performed to compare serial data (follicle size and follicle number changes over the day), and ANOVA or paired t-test was used for single-point numerical measurements. Count data were compared using Fisher's Exact test. Synchronicity of wave appearance was compared using Barlett and Levene's test for homogeneity of variance.

[0222] Results: The results of Example 3 are presented in Tables 3-6.

[0223] Table 3: Days of wave appearance after no treatment (control period) or after a single i / m injection of 1.5 mg cetrorelix (treatment period).

[0224]

[0225]

[0226] The emergence of the next wave occurred on average 5.33 ± 0.70 days after a single treatment with 1.5 mg cetrorelix on the random day of the wave, compared to 8.93 ± 1.21 days without treatment (control period).

[0227] Table 4: Statistical analysis of the next wave after cetrorelix treatment and untreated controls.

[0228]

[0229] Table 5: Bartlett's test for homogeneity of variance of durations.

[0230] source DF Chi-square Pr>ChiSq Group 1 3.8654 0.0493

[0231] Table 6: Levene's test for homogeneity of variance ANOVA of duration squared deviations from the group mean.

[0232] source DF sum of squares Mean Square F-number Pr>F Group 1 1409.0 1409.0 5.65 0.0245 error 28 6980.6 249.3

[0233] The change in duration after cetrorelix treatment (variance 7.38) was lower than that in the control period (22.067), indicating synchronization of wave appearance [Barlett's test for homogeneity of variance p = 0.049, Levene's test p = 0.025].

[0234] The CIs after cetrorelix treatment ranged from 3.8 to 6.8 days compared with control CIs of 6.3 to 11.5 days.

[0235] Example 4: Effects of Cetrorelix on Preselection, Growth and Fate of Degenerating Dominant Follicles in Beef Heifers

[0236] Specific objectives: The primary objective was to determine the growth pattern of dominant and first subordinate follicles in cattle after treatment with the GnRH antagonist cetrorelix during the preselection (days 1-2), growth (days 3-4), and late quiescence / early involution phases (days 6-7) of the follicular wave. Secondary objectives were to document changes in CL function and progesterone production.

[0237] Hypothesis: Treatment with a GnRH antagonist results in loss of follicular dominance and the emergence of new waves of follicles at consistent and predictable intervals following treatment, regardless of the follicular stage at the time of treatment (i.e., preselection, growth, late quiescent / early involution phase of the dominant follicle).

[0238] Experimental Design: A schematic diagram of the experimental design is shown in Figure 4In. This study was conducted on Hereford crossbred beef heifers (n=32) in puberty at the U.Sask.Livestock and Forage Centre of Excellence (LFCE) of the University of Saskatchewan. An initial transrectal ultrasound examination was performed to confirm puberty by the presence or absence of a corpus luteum (CL). The animals were also weighed at this time. An intramuscular (im) injection of a prostaglandin F2a analog (cloprostenol) was administered to animals with functional CL to cause CL regression and ovulation. Daily ultrasound examinations were performed to detect ovulation and record the number, size, and position of follicles and CL in both ovaries. The day of follicular wave appearance was initially estimated by a sudden increase in the number of 3-4mm follicles, and was retrospectively confirmed as the day when a dominant follicle of 4-5mm in size was first detected (defined as day 0). Follicle and hormone data from all animals were collected at a complete interovulatory interval (endpoints are shown in the next section).

[0239] Table 7: Treatment regimen (all treatments were given intramuscularly).

[0240]

[0241] * Cetrorelix was given intramuscularly in the neck as 3 ml of 0.5 mg / ml cetrorelix acetate in 5% D-mannitol aqueous solution.

[0242] Animals were randomly divided into 4 groups according to the day of ovulation. Group 1 (control group) heifers were given 3 mL of saline (negative control for sham treatment; n=3 on days 1 and 2, n=3 on days 3 and 4, and n=2 on days 6 and 7). Group 2 heifers were given 1.5 mg of cetrorelix on days 1 and 2 (preselected dominant follicle phase, n=8), Group 3 heifers were given cetrorelix on days 3 and 4 (dominant follicle growth phase, n=8), and Group 4 heifers were given cetrorelix on days 6 and 7 (dominant follicle late quiescence / early involution phase, n=8). The dose of cetrorelix (1.5 mg per treatment), route of administration (intramuscular), and duration of treatment (i.e., 2 doses separated by 24 hr) were selected based on Example 2 and previous studies in cattle (Ulker et al., 2001).

[0243] From initial ovulation to the first post-treatment ovulation, changes in the size of the dominant follicle, the largest subordinate follicle, and the CL, as well as the number of follicles in the 3-5, 6-8, and 9+ mm size categories, were recorded daily. The interwave interval was determined based on the duration between the appearance of consecutive follicular waves.

[0244] Blood samples were also collected every 12 hr by jugular vein puncture for measurement of plasma LH concentrations from -36 hr to +72 hr of treatment. Blood samples were collected daily from 1 day before treatment to 7 days after treatment for measurement of plasma progesterone concentrations. ProcMixed repeated measures ANOVA (SAS Version 9.4) was performed to compare serial data (follicle size, number of follicles, CL size, plasma LH, plasma progesterone).

[0245] Results: In the groups treated with cetrorelix on days 1-2 and 3-4, the dominant follicles stopped growing ( Figure 5 ). The size of the dominant follicle in the untreated control group indicated that the follicle had entered a quiescent phase by Day 6-7, i.e., no further increase in diameter occurred after Day 6-7. Therefore, no difference in the diameter of the dominant follicle was expected between heifers treated with Cetrorelix on Days 6-7 compared to untreated control heifers.

[0246] Table 8: Follicle dynamics after cetrorelix treatment.

[0247]

[0248] There was a significant difference in the diameter of the dominant follicle between the control group and the groups on days 1-2 and 3-4 (Table 8). This suggests that cetrorelix causes early regression of the follicle. There was no difference between the control and day 6-7, as the dominant follicle was already ready to start regression.

[0249] Table 9: Synchronicity of wave appearance after cetrorelix treatment.

[0250]

[0251] The data from Table 9 indicate that wave emergence occurred 5.3 ± 0.3 days after the first cetrorelix treatment, regardless of the stage of the dominant follicle. These results indicate a high degree of synchronization of wave emergence between groups.

[0252] Table 10: Follicular wave length during and after treatment with Cetrorelix.

[0253]

[0254] The day 1-2 group had significantly shorter inter-wave intervals. There were no differences in the length of the ovulatory interval or wave 2. This supports that cetrorelix has a short half-life and does not continue to affect later waves.

[0255] Table 11: Corpus luteum dynamics after cetrorelix treatment.

[0256]

[0257] The maximum CL diameter in the day 1-2 group was smaller than that in the control group. One animal in this group had a transient CL. The time when the CL diameter was maximum was not affected by cetrorelix treatment

[0258] In summary, the following conclusions can be drawn from this embodiment:

[0259] i. Cetrorelix causes degeneration of the dominant follicle regardless of growth phase status

[0260] ii. A new wave appeared 5.3±0.3 days after the first injection of cetrorelix. There was a high degree of synchronization

[0261] iii. The next wave, ovulation and cycle length are not affected

[0262] iv. The next wave of dominant follicles is ready to ovulate within 9 days

[0263] Example 5: Single injection of cetrorelix induces new follicular waves in heifers

[0264] Primary objective: To test whether a single 3 mg i / m cetrorelix injection given to heifers several days after the emergence of follicular waves (ie, at different states of the dominant follicle) would induce the emergence of synchronized waves.

[0265] Hypothesis: 1) A single Cetrorelix injection will induce regression of the dominant follicle. New waves will emerge at consistent times. 2) A high degree of ovulatory synchrony will be achieved after PGF treatment. 3) Preovulatory follicles from the induced wave will be developmentally competent

[0266] Methods: A schematic diagram of the experimental design of Example 5 is shown in Figure 6 middle.

[0267] On random days of the cycle, heifers were treated with two i / m injections of prostaglandin analogs and the ovulation day (defined as day 0 of the wave) was observed by transrectal ultrasonography. The heifers were then randomly divided into 4 groups (n=7 to 8 per group) and treated with saline (control group) or a single 3 mg i / m injection of cetrorelix on day 1, day 3, or day 6 of the first follicular wave. Nine days after saline or cetrorelix treatment, heifers were given prostaglandin analog injections and artificial insemination was performed when the heifers showed estrus behavior (detected by tail paint). Pregnancy diagnosis was performed 30 days after artificial insemination.

[0268] Results: The dominant follicles during the treatment wave exhibited a growth pattern similar to those treated in Example 4. Two days after cetrorelix treatment, the dominant follicles began to regress ( Figure 7 , left panel). The growth pattern of the dominant follicle in the next (second) wave ( Figure 7 , right panel) is not affected by previous processing.

[0269] Table 12: Synchronicity of follicular wave emergence after a single injection of Cetrorelix Wave emergence occurred 3.5 days after Cetrorelix treatment regardless of the stage of the dominant follicle.

[0270]

[0271] Table 13: Ovulatory follicle and CL size after a single i / m injection of cetrorelix.

[0272]

[0273] Table 14: Pregnancy rates following cetrorelix treatment. Overall pregnancy rates were similar between cetrorelix treated cattle (n=12 / 21, 57%; three cetrorelix groups combined) and saline treated controls (n=3 / 5=60%).

[0274]

[0275] The following conclusions are drawn from this example:

[0276] i. A new wave appeared 3.5 ± 0.4 days after a single 3 mg cetrorelix i / m treatment. The status of the dominant follicle and the follicular wave had no effect on the interval between treatment and wave appearance.

[0277] ii. There is a high degree of synchronization between the occurrence of the wave and ovulation.

[0278] iii. Oocytes produced in the second (induced) wave are fertile.

[0279] Example 6: Effects of Cetrorelix during the Luteal Phase (High Progesterone) and Preovulatory Phase (Low Progesterone) in Heifers

[0280] Specific objective: To compare the growth pattern and ovulatory potential of the dominant follicle in cattle treated with cetrorelix during the luteal phase (days 5-6 of the wave during the functional luteal phase) and the preovulatory phase (days 5-6 of the wave during the luteal regression phase).

[0281] Hypothesis: 1. Cetrorelix will cause regression of both mid-quiescent dominant follicles and preovulatory stage dominant follicles, and 2. Dominant follicles in animals treated with Cetrorelix will not ovulate in response to exogenous LH (hCG).

[0282] Experimental Design: This study was conducted on pubertal Hereford crossbred beef heifers (n=24) at the University of Saskatchewan Livestock and Forage Excellence Center (LFCE) using a 2×2 factorial design (Table 15). Initial transrectal ultrasonography was performed to confirm post-pubertal or postpartum cyclicity by the presence or absence of a corpus luteum (CL). Animals with CL were weighed and treated with prostaglandin F2a analogs as in Example 4 and monitored by ultrasonography to detect ovulation. The day of ovulation was considered the day of wave occurrence (defined as day 0).

[0283] Table 15: Experimental design (2×2 factorial design)

[0284]

[0285] On the day of ovulation, heifers were randomly divided into 4 groups. Those in the preovulatory group were given prostaglandins on days 4 and 4.5 of the wave, while those in the luteal group were allowed to maintain CL (i.e., no prostaglandin treatment). As shown in Tables 16 and Figure 8 Treatments are given as outlined in the schematic timeline of . The cetrorelix group was given 3 mg cetrorelix i / m on day 5. All animals were treated with hCG (Chorulon, 1500 IU, im) on day 7 of the wave (i.e., 48 hr after cetrorelix / saline treatment) to determine the ovulatory potential of the dominant follicle.

[0286] Table 16: Treatment options:

[0287]

[0288] Blood samples were collected 24 hr after treatment with or without cetrorelix (control group) for measurement of plasma concentrations of LH and progesterone. The data were analyzed in a similar manner to Example 4.

[0289] Results: All 4 groups (cetrorelix and control; low and high progesterone) ovulated after treatment with hCG, indicating that, in contrast to the proposed hypothesis 2, the dominant follicles in the high progesterone environment and the preovulatory follicles in the low progesterone environment maintained ovulatory potential for at least 48 hours after cetrorelix treatment and had not yet regressed, i.e., the follicles responded to exogenous hCG (LH) by ovulation. Cetrorelix heifers ovulated on day 9 or 10 (3 to 4 days after cetrorelix treatment), resulting in the initiation of a new follicular wave. This duration matches previous experiments, indicating that cetrorelix treatment followed by LH or hCG may also be an effective way to initiate the emergence of synchronized follicular waves.

[0290] Example 7: Effects of a single injection of cetrorelix given on a random day of the follicular wave (and estrous cycle) in heifers

[0291] Primary Objective: To examine the effects of cetrorelix treatment given on random days of the cycle on follicular wave synchronization, ovulation synchronization after fixed-time artificial insemination, and pregnancy rates in heifers

[0292] Hypotheses: 1) Cetrorelix treatment given on random days of the cycle will induce new waves at consistent times, 2) the Cetrorelix regimen results in ovulation synchronization and acceptable pregnancy rates after fixed-time artificial insemination, and 3) this steroid-free ovulation synchronization regimen is as effective as an estradiol-based fixed-time AI regimen.

[0293] Methods: A schematic diagram of the experimental design of Example 7 is shown in Fig. 9 middle.

[0294] The experiment was conducted on 38 mature Angus heifers at the Federal University of Santa Maria, Brazil. Heifers on a random day of the cycle (defined as day 0) were divided into 2 groups (n=19 per group) and treated with a single i / m injection of 3 mg of cetrorelix or estradiol benzoate (1 mg i / m). A progesterone-releasing device (CIDR) was placed in the vagina of the heifer at this time. Eight days after cetrorelix or estradiol treatment, the heifers were given a 500 μg i / m injection of cloprostenol (prostaglandin analog) and the CIDR device was removed. Artificial insemination (AI) was performed when the heifers showed estrus behavior (detected by tail paint). Heifers were given a 250 μg i / m injection of gonadorelin acetate (GnRH analog) at the time of artificial insemination. If ovulation did not occur within 24 hrs of GnRH treatment, a second AI was performed. Pregnancy diagnosis was performed 29-30 days after artificial insemination.

[0295] Results: A greater proportion of heifers showed estrus behavior after cetrorelix treatment compared with estradiol-progesterone treatment ( Fig.10 , left panel). In both treatment groups, most ovulations occurred between 72 and 96 hours after CIDR removal ( Fig.10 , right panel).

[0296] Table 17: Pregnancy rates following cetrorelix treatment compared to estradiol-progesterone regimen. Early ovulation was detected at the time of artificial insemination in two heifers in the cetrorelix group and two heifers in the estradiol-progesterone group.

[0297]

[0298]

[0299] The cetrorelix regimen resulted in synchronized ovulation and numerically higher pregnancy rates (79%) in Angus heifers following fixed-time artificial insemination than the current standard estradiol-progesterone regimen (68%); however, there were no statistical differences between the two treatments. Pregnancy rates did not differ between heifers inseminated once or requiring a second insemination.

[0300] The following are the conclusions of this study:

[0301] i. Based on ovulation and pregnancy data, treatment with the GnRH antagonist cetrorelix results in regression of the dominant follicle regardless of the status of the follicular wave

[0302] ii. Based on the ovulation and pregnancy data, cetrorelix treatment resulted in the appearance of new follicle waves at consistent intervals from the start of treatment.

[0303] iii. In the Cetrorelix regimen, the majority (90%) of ovulations occurred between 72 and 96 hrs after prostaglandin injection, and heifers exhibited improved estrus behavior following the Cetrorelix regimen compared to the estradiol-progesterone based regimen.

[0304] iv. The pregnancy rate of beef heifers after the cetrorelix regimen (79%) was slightly better than the pregnancy rate after the estradiol-progesterone regimen (68%), but there was no statistical difference.

[0305] Example 8: Field Trial to Determine Conception (Pregnancy) Rates in Postpartum Beef Cows Following Single Injection of Cetrorelix Administered on Random Days of the Follicular Wave and Estrous Cycle

[0306] Objective: The objective of this study was to compare conception rates after a single fixed-time artificial insemination in multiparous cows using cetrorelix versus an estradiol synchronization protocol.

[0307] Methods: A schematic diagram of the experimental design of Example 8 is shown in Fig.11 middle.

[0308] The field trial was conducted by faculty from the Federal University of Santa Maria on a client beef farm in Brazil in December 2022. A total of 216 Brahman x Angus crossbred beef postpartum cows were used in this experiment. The cows were divided into two treatment groups (n = 106 per group) and given cetrorelix 3mg i / m or estradiol benzoate on a random day of the cycle (defined as day 0). An intravaginal progesterone device (CIDR) was placed in all cows on day 0. The CIDR device was removed on day 8, and the animals were given prostaglandin F2a analogs and equine chorionic gonadotropin (eCG). Fixed-time artificial insemination was performed 54hr after CIDR removal, and the animals were given GnRH at this time. Pregnancy diagnosis was performed on day 30 after artificial insemination. Transrectal ultrasound examinations were performed on days 0, 8, 10, and 40.

[0309] Results: The results of the study are shown in Tables 18 and 19.

[0310] Table 18. Size of dominant follicles on the day of CIDR removal (day 8) and fixed-time artificial insemination (day 10)

[0311] Day 8 (PGF) Day 10 (Al) Cetrorelix 11.04±0.48 12.72±0.65 Estradiol benzoate 8.60±0.39 11.38±0.56

[0312] On the day of prostaglandin treatment (CIDR removal day) and fixed-time artificial insemination day, the dominant follicles in the Cetrorelix group were larger than those in the estradiol group. The mucus secretion and estrus behavior of cows in the Cetrorelix group were higher than those in the estradiol group.

[0313] Table 19. Pregnancy rate 30 days after fixed-time artificial insemination.

[0314] Pregnancy / AI percentage% Cetrorelix 46 / 108* 42.6％ Estradiol benzoate 48 / 106* 45.6％

[0315] * Chi-square P value = 0.69

[0316] There was no difference in conception (pregnancy) rates between the cetrorelix regimen (42.6%) and the estradiol-based regimen (45.6%). Of note, on the day of fixed-time artificial insemination, the ambient temperature exceeded 40°C, resulting in extreme heat stress, which may have affected the overall pregnancy rates in both groups. However, there was no difference in pregnancy rates between the two groups, indicating that the cetrorelix regimen was as effective as the estradiol regimen even under extreme heat stress conditions.

[0317] Conclusion: The overall conclusions of this study are:

[0318] i. On the day of CIDR removal and fixed-time artificial insemination, the dominant preovulatory follicle in the cetrorelix group had a size advantage over the estradiol-based regimen.

[0319] ii. When comparing pregnancy rates, the cetrorelix regimen was as effective as the estradiol-based regimen (i.e., there was no difference in conception rate between the cetrorelix-based regimen and the estradiol-based regimen). Cows on the cetrorelix regimen showed better estrus behavior.

[0320] Example 9: Effects of four GnRH antagonists on LH secretion in bovine pituitary cells in vitro

[0321] Objective: To compare the efficacy of four GnRH antagonists (cetrorelix, degarelix, abarelix, and relugolix) in blocking GnRH-mediated LH release from bovine gonadotrophs using in vitro cultures of bovine pituitary glands.

[0322] Hypothesis: Pituitary cells treated in vitro with a GnRH antagonist will have lower levels of LH secretion than untreated controls following stimulation of the cells with GnRH.

[0323] Methods: The heads of mature non-pregnant cows (n=4) were collected from a local slaughterhouse, and the pituitary glands were dissected within 60 minutes after slaughter. After dissection, the pituitary was immediately immersed in ice-cold collection medium. The pituitary was washed 3 times in the collection medium, and the neurohypophysis was dissected from the adenohypophysis. Using a scalpel, the adenohypophysis (anterior pituitary) was cut into 1mm×1mm blocks and transferred to a 50mL falcon tube containing DMEM medium with 0.5% BSA. The tissue blocks were washed by manually stirring the tube for 1 minute. The tissue blocks were allowed to settle to the bottom of the tube, and the supernatant was discarded. The macerated blocks were transferred to a 15mL Falcon tube with dissociation medium and incubated at 37°C for 60 minutes. The tube was vortexed for 1 minute every 10 minutes to ensure complete dissociation of the tissue. The dissociation medium was inactivated using DMEM containing 10% SFB, and the tube was then manually stirred for 1 minute. The dissociated cells were placed in a 40 μm cell strainer to remove undigested tissue and placed in two 15 mL Falcon tubes. The cells were centrifuged at 200 × g for 10 minutes and the supernatant was discarded. The cells were washed three times by resuspending in a DMEM solution with red blood cell lysis buffer, followed by centrifugation at 200 × g for 10 minutes. The cells were then washed with a DMEM solution and centrifuged at 200 × g for 10 minutes. After the last centrifugation, the cells were resuspended in DMEM and the trypan blue test was used to determine the cell count and assess viability. DMEM containing 10% fetal bovine serum (FBS) was treated with 10% activated charcoal-dextran and passed through a 0.2 μm filter. The cells were then plated at 1 × 10 6 The cells were plated at a concentration of 100 viable cells in a 24-well tissue culture plate and incubated for 24 hours at 37° C. The next day, the medium was replaced with DMEM without FBS to starve the cells and incubated for another 24 hours.

[0324] Treatment: Based on the results of preliminary studies, the selected concentration of cetrorelix (cetrorelix acetate) was 300 μg. Cetrorelix was dissolved in trace levels of DMSO and then dissolved in ultrapure water. To standardize the treatment, the nanomolar concentration of each GnRH antagonist was matched to the nanomolar concentration of cetrorelix. The final concentrations used for further experiments were: cetrorelix 300 μg, degarelix (degarelix acetate) 340 μg, and abarelix (abarelix acetate) 300 μg. Both degarelix and abarelix were dissolved in ultrapure water. The equivalent nanomolar concentration of relugolix dissolved in methanol was 130 μg. Treated cells were pre-incubated with cetrorelix, degarelix, relugolix or abarelix for 60 minutes. After pre-incubation, cells were incubated with 1 nM or 10 nM GnRH (gonadorelin, Fertagyl, Merck Animal Health) for 30 minutes. GnRH dosage was based on pituitary studies by Paolicchi et al. 1999). The culture medium was collected and centrifuged at 200×g for 10 minutes to remove cells. Positive (control) groups were treated with 1 nM and 10 nM GnRH alone to determine maximal LH secretion. LH concentration in the culture medium was measured by a commercial bovine-specific ELISA kit and reported as mIU / ml culture medium.

[0325] Results: The dissociation procedure yielded 20-50 × 10 per pituitary gland. 6 cells (n=4) and had >80% cell viability. It is noteworthy that cetrorelix is ​​a fast-acting peptide, relugolix is ​​a fast-acting non-peptide, and degarelix is ​​a slow-acting peptide. The results of the study are shown in Fig.12 and Fig.13 and are shown in Tables 20 and 21.

[0326] Table 20: LH concentration in culture medium from in vitro pituitary cells when challenged with 1 nM GnRH

[0327]

[0328] Table 21: LH concentration in culture medium from in vitro pituitary cells when stimulated with 10 nM GnRH.

[0329]

[0330] It is expected that LH secretion will be reduced compared to the positive control. Cetrorelix was most effective in blocking GnRH-induced LH secretion under both 1 nM GnRH and 10 nM GnRH stimulation, i.e. Fig.12 and Fig.13As well as the cetrorelix group in Tables 20 and 21 had the lowest LH levels for each GnRH concentration given. Both degarelix and relugolix showed some degree of LH suppression as they had LH levels lower than the control. At the concentrations used, abarelix had no effect on LH suppression under 1 nM GnRH challenge, but caused a modest decrease in LH concentrations when challenged with 10 nM LH. When the results of 1 nM GnRH and 10 nM GnRH challenge were compared between the same drugs, a dose-dependent increase in LH secretion was recorded. In addition, the ranking of the drugs remained unchanged between 1 nM and 10 nM GnRH challenge.

[0331] Conclusion: The overall conclusions of this study are:

[0332] i. Cetrorelix is ​​a fast-acting GnRH antagonist peptide, relugolix is ​​a fast-acting non-peptide, and degarelix is ​​a slow-acting peptide. In in vitro incubations, all three GnRH antagonists resulted in a blockade of GnRH-induced LH secretion compared to controls at low (1 nM) and high (10 nM) doses of GnRH.

[0333] ii. Abarelix failed to block LH secretion at 1 nM GnRH challenge, but showed some modest blocking effect at 10 nM GnRH challenge. Abarelix may require higher nanomolar concentrations to effectively reduce LH levels.

[0334] iii. Cetrorelix is ​​the most effective antagonist in reducing LH levels.

[0335] iv. GnRH antagonists differ in their blocking efficiency. The similar ranking of cetrorelix, degarelix, relugolix, and abarelix at 1 nM and 10 nM GnRH challenge suggests consistency in responses.

[0336] v. The results of this experiment confirm that the underlying mechanism of action (i.e., the ability to block LH secretion from the pituitary gland) is similar between the four GnRH antagonists tested, but their blocking efficiency at a fixed nanomolar concentration varies between the drugs. Therefore, based on these results, degarelix, relugolix, and abarelix, as well as other GnRH antagonists, are predicted to have similar in vivo effects as shown for cetrorelix, such as synchronization of follicular wave emergence, however, each drug will require calibration of the effective dose.

[0337] Example 10: Effects of four GnRH antagonists (cetrorelix, degarelix, relugolix, abarelix) on GnRH-induced plasma LH levels and ovulation in cattle

[0338] Objective: To identify the effects of GnRH antagonists on plasma LH levels and ovulation in heifers injected with GnRH.

[0339] Hypothesis: 1) GnRH antagonists (cetrorelix, degarelix, relugolix, abarelix) will prevent the LH rise regardless of whether GnRH is administered. 2) Animals treated with GnRH antagonists will not ovulate.

[0340] Methods: Estrus heifers (n=10) with functional CL were selected at the Federal University of Santa Maria. Heifers were administered intramuscular (i / m) injections of prostaglandin F2a analogs (PGF; Lutalyse 5 ml or Estrumate 2 ml) to induce CL regression. Heifers were randomly placed into 4 treatment groups at the time of PGF injection: Trt1 (n=2, 3 mg Abarelix); Trt2 (n=2, 3 mg Degarelix), Trt3 (n=2, 3 mg Relugolix) and Trt 4 (n=4; Cetrorelix 3 mg). Heifers were treated with these GnRH antagonists 1.5 days (36 hr) after PGF injection, and the GnRH antagonist treatment time point was defined as -60 minutes. Heifers were treated with GnRH (2 mg Fertagyl i / m) at 0 min to induce ovulation. Jugular puncture plasma samples were collected every 60 minutes for 3 hours (from -60 minutes to 120 minutes, when GnRH was administered, t = 0 minutes). Plasma samples were kept at 4°C overnight and analyzed for LH the next day using a bovine-specific ELISA commercial kit. Transrectal ultrasonography was performed at the time of GnRH injection (0 hr) and 2 days later (48 hr after GnRH injection) to record the size of the dominant follicle and detect ovulation after treatment.

[0341] result: Fig.14 A shows a proof of concept example using Cetrorelix. GnRH injection given at 0 min: untreated controls showed a 1.5-fold increase in plasma LH concentrations within 30 min, while LH levels remained unchanged in heifers pretreated with Cetrorelix 60 min prior to GnRH injection. Table 22 shows plasma LH concentrations for individual animals at -60 hr (GnRH antagonist injection time), 0 min (GnRH challenge), and 60 min and 120 min after GnRH injection.

[0342] Table 22: Plasma LH concentrations (mIU / ml) following GnRH challenge in heifers pretreated with abarelix, degarelix, relugolix, cetrorelix, and control (no pretreatment). Data from individual heifers and mean ± standard error for each group are presented. Time 0 min = time of GnRH injection. Injection of GnRH antagonist was given 60 min prior to GnRH injection (at time -60 min).

[0343]

[0344]

[0345] Due to the large individual variability in plasma LH concentrations before treatment with LH antagonists in Table 22, the data were normalized to the time of GnRH injection (considered as 1), and the fold change for each group is presented in Fig.14 In B. In the positive control group (i.e., without any GnRH antagonist treatment), LH concentration increased 1.57-fold within 2 hours, while animals pretreated with abarelix, degarelix, relugolix or cetrorelix did not record an increase in LH when challenged with GnRH, indicating that all four tested GnRH antagonist drugs effectively blocked GnRH-induced LH secretion in vivo. Table 23 shows the size of the dominant follicle at the time of GnRH treatment and 48 hr later, and at 48 hr after GnRH treatment when ovulation was detected. All cows pretreated with abarelix, degarelix, relugolix or cetrorelix failed to ovulate 48 hr after GnRH injection, while the positive control group animals (i.e., those given GnRH alone) all ovulated at 48 hr.

[0346] Table 23. Dominant follicle diameters of individual heifers in each group at and 48 hrs after GnRH treatment. The last column lists whether ovulation occurred within 48 hrs of treatment (yes / no).

[0347]

[0348]

[0349] Conclusion: Both hypotheses were supported. All four GnRH antagonists (abarelix, degarelix, relugolix, and cetrorelix) blocked GnRH-induced LH release and prevented GnRH-induced ovulation in cattle, suggesting that any of these GnRH antagonist drugs could be used in the breeding management program described herein.

[0350] Example 11: Effects of GnRH antagonists on follicular dynamics in cows

[0351] Objective: To test the effect of other GnRH antagonists (selected drugs include but are not limited to degarelix, abarelix, ganirelix, antitide, relugolix, elagolix, or acyline) on the development of follicular waves when injected on days 1 and 2, days 3 and 4, or days 5 and 6 after the onset of the follicular wave.

[0352] Methods: A schematic diagram of the experimental design of Example 11 is shown in Fig.15 middle.

[0353] The experimental design was very similar to Example 4, except that PGF was used to induce ovulation before the start of the experiment, and FA (follicle ablation) was performed 7 days after PGF (approximately 3-4 days after ovulation).

[0354] Treatment will be done with two i / m injections (24 hrs apart) - one or more of the following drugs will be tested in parallel: degarelix, abarelix, ganirelix, antitide, relugolix, elagolix or acyline. Treatments will be given on days 1-2, or days 3-4 or days 5-6 of the wave.

[0355] PGF was given on day 4 of the new (post-treatment) wave to induce ovulation for reuse of animals or for breeding (ie, not part of the experimental design).

[0356] The following endpoints will be measured:

[0357] Daily measurement of the diameter of the largest (dominant) follicle present at the time of treatment administration (until involution or ovulation)

[0358] New wave appears on the day after processing

[0359] Daily diameter measurement of the new wave of dominant follicles after treatment

[0360] Ovulation day after treatment

[0361] Ovulation day after the wave appears

[0362] Daily CL diameter

[0363] Doppler echotexture of the CL (visual scoring from 1 to 4): 1 = functional estrous CL at the time of PGF injection; 2 = 24 hr after PGF; 3 = 48-72 hr after PGF; 4 = one day after ovulation detection (metestrus). CL diameter was also used in conjunction with the grading scheme.

[0364] Progesterone in blood samples 48 hours after GnRH antagonist treatment

[0365] Expected results: Synchronization of follicular waves by the GnRH antagonists tested.

[0366] Example 12: Comparison of different GnRH antagonists

[0367] Objective: To compare the effects of different GnRH antagonists on follicular dynamics in cows

[0368] The effects of other GnRH antagonists (e.g., acyline, anrelix / teverelix, degarelix, ganirelix, antitide, relugolix, elagolix, abarelix, prarelix, ramorelix, antitide, detirelix, ozarelix, linzagolix, opigolix, sufgolix, and / or A-75998) were tested in parallel for comparison with cetrorelix.

[0369] Methods: A schematic diagram of the experimental design of Example 12 is shown in Fig.16 The experimental design and endpoints were very similar to Example 11, except that only one administration time point (day 3-4 of the wave) of drug treatment was tested.

[0370] Expected Results: Cetrorelix, acyline, anrelix / teverelix, degarelix, ganirelix, antitide, relugolix, elagolix, abarelix, prarelix, ramorelix, antitide, detirelix, ozarelix, linzagolix, opigolix, sufgolix, and / or A-75998 all synchronize the emergence of follicular waves, but the duration between treatment and the day of FWE may vary between the drugs tested.

[0371] Example 13: Comparison of single dose and double dose

[0372] Objective: To compare the effects of a single versus two injections (24 hrs apart) of treatment (cetrorelix vs. degarelix / other GnRH antagonists) on follicular dynamics in heifers.

[0373] Methods: A schematic diagram of the experimental design of Example 13 is shown in Fig.17 The experimental design and endpoints were very similar to Example 11, except that the number of administrations of the GnRH antagonist (once vs. twice) given on day 3 or day 3-4, respectively, was compared.

[0374] Expected Results: Administration of a single dose or two-dose series of GnRH antagonists synchronizes FWE, but the duration between treatment and FWE day may vary.

[0375] Example 14: Optimization of Cetrorelix Dosage in (Beef) Cattle

[0376] Objective: To determine the minimum effective i / m dose of a single injection of cetrorelix to synchronize wave appearance with ovulation.

[0377] Methods: A schematic diagram of the experimental design of Example 14 is shown in Fig.18Lactating beef cows on a random day of the cycle (defined as day 0) were divided into 3 groups (n=15 per group) and treated with a single i / m injection of 1) 3 mg cetrorelix, 2) 2 mg cetrorelix, or 3) 1 mg cetrorelix. A progesterone-releasing device (CIDR) was placed in the vagina of the heifer at this time. Eight days after cetrorelix treatment, the cows were given a prostaglandin analog injection and the CIDR device was removed. The animals were tail-painted or Estrotect patched when the CIDR was removed and checked every day until ovulation. Fixed-time artificial insemination was performed 54 hr after CIDR removal, and the animals were given GnRH at this time. Pregnancy diagnosis was performed 30 and 60 days after artificial insemination.

[0378] The following endpoints were measured:

[0379] Duration between cetrorelix injection and wave appearance

[0380] Synchronicity of waves

[0381] Diameter of dominant follicle during PGF and AI

[0382] Duration between Cetrorelix and ovulation

[0383] Ovulation rate and ovulation synchrony

[0384] Luteal blood flow and plasma progesterone on day 7 after ovulation

[0385] Plasma progesterone levels on the day of cetrorelix treatment, PGF, and AI

[0386] Pregnancy rates at 30 and 60 days after AI

[0387] Expected results: The minimum effective i / m dose of cetrorelix is ​​1 mg to 2 mg.

[0388] Although the present application has been described with reference to what are currently considered to be preferred embodiments, it should be understood that the present application is not limited to the disclosed embodiments. On the contrary, the present application is intended to cover various modifications and equivalent arrangements included in the spirit and scope of the appended claims. The scope of the claims should not be limited by the preferred embodiments and embodiments, but should be given the broadest interpretation consistent with the specification as a whole.

[0389] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0390] Table 24: Suitable GnRH antagonists.

[0391]

[0392]

[0393]

[0394]

[0395] sequence

[0396] SEQ ID NO: 1 (human kissin-10)

[0397] YNWNSFGLRF

[0398] SEQ ID NO:2 (Cetrelix-synthetic peptide)

[0399] X1X2X3SYX4LRPX5

[0400] wherein X1 = N-acetyl-3-(2-naphthyl)-D-alanine; X2 = 4-chloro-D-phenylalanine; X3 = 3-(3-pyridyl)-D-alanine; X4 = D-citrulline; and X5 = D-alanine

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Claims

1. A non-therapeutic method for synchronizing follicular wave emergence (FWE) in a population of female mammals, the method comprising: An effective amount of a GnRH antagonist is administered to each mammal in the population of mammals.

2. A non-therapeutic method for synchronizing ovulation in a population of female mammals, the method comprising: I) a) administering an effective amount of a GnRH antagonist to each mammal of said population of female mammals; as well as b) administering an effective amount of an ovulation inducing agent to each mammal of said population of female mammals at a fixed time after said administration of said GnRH antagonist; or II) a) administering to each mammal of said population of female mammals an effective amount of a GnRH antagonist and an effective amount of a progesterone or a progesterone analog; b) stopping the administration of said progesterone or progesterone analog at a fixed time after said administration of said GnRH antagonist and administering an effective amount of prostaglandin (PGF) or a PGF analog; as well as c) administering an effective amount of an ovulation inducing agent to each mammal of said population of female mammals at a fixed time after cessation of progesterone or a progesterone analog and / or administration of said PGF or a PGF analog.

3. The method of claim 1 or claim 2, wherein the GnRH antagonist comprises cetrorelix, acyline, anrelix / teverelix, degarelix, ganirelix, antitide, relugolix, elagolix, abarelix, prarelix, ramorelix, antitide, detirelix, ozarelix, linzagolix, opigolix, sufgolix, and A-75998.

4. The method of claim 3, wherein the GnRH antagonist comprises cetrorelix.

5. The method of claim 3, wherein the GnRH antagonist comprises abarelix.

6. The method of claim 3, wherein the GnRH antagonist comprises degarelix.

7. The method of claim 3, wherein the GnRH antagonist comprises relugolix.

8. The method according to any one of claims 1 to 7, wherein the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

9. The method according to any one of claims 1 to 8, wherein said administering of said GnRH antagonist comprises at least one treatment, optionally 2 or 3 treatments.

10. The method of any one of claims 1 to 9, wherein the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of about 5 μg / kg body weight to about 200 μg / kg body weight, optionally at a dose of about 20 μg / kg body weight or about 40 μg / kg body weight.

11. The method of any one of claims 1 to 7, wherein the GnRH antagonist is administered via a vaginal drug release device.

12. The method according to any one of claims 2 to 11, wherein the progesterone or progesterone analogue is administered via a vaginal drug release device.

13. The method according to any one of claims 1 to 12, wherein the mammal is livestock, optionally the livestock is bovine, camelid, equine, porcine, ovine or caprine.

14. The method of any one of claims 1 to 12, wherein the mammal is a wild animal, optionally a bison, elk, reindeer, deer, musk ox, non-human primate, canine or feline, optionally a tiger, lion or panther.

15. The method according to any one of claims 1 to 14, wherein the population is a mixed population.

16. The method according to any one of claims 1 to 15, further comprising artificially inseminating each mammal in the female mammal population at a fixed time after administration of the GnRH antagonist, and optionally collecting one or more embryos from each mammal in the female mammal population at a fixed time after artificial insemination.

17. The method according to any one of claims 1 to 15, further comprising mating each mammal in the female mammal population at a fixed time after administration of the GnRH antagonist, and optionally collecting one or more embryos from each mammal in the female mammal population at a fixed time after mating.

18. The method of any one of claims 1 to 15, further comprising transferring an embryo to each animal of the population of female mammals at a fixed time after administration of the GnRH antagonist.

19. A non-therapeutic method of inducing follicular wave emergence (FWE) in a female mammal, the method comprising: An effective amount of a GnRH antagonist is administered to the female mammal.

20. The method of claim 19, wherein the GnRH antagonist comprises cetrorelix, abarelix, degarelix, ganirelix, anrelix / teverelix, prarelix, ramorelix, antitide, detirelix, ozarelix, acyline, elagolix, linzaglix, relugolix, opigolix, sufgolix, or A-75998.

21. The method of claim 20, wherein the GnRH antagonist comprises cetrorelix.

22. The method of claim 20, wherein the GnRH antagonist comprises abarelix.

23. The method of claim 20, wherein the GnRH antagonist comprises degarelix.

24. The method of claim 20, wherein the GnRH antagonist comprises relugolix.

25. The method according to any one of claims 19 to 24, wherein the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

26. The method according to any one of claims 19 to 25, wherein said administering of said GnRH antagonist comprises at least one treatment, optionally 2 or 3 treatments.

27. The method of any one of claims 19 to 26, wherein the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of about 20 μg / kg body weight to about 40 μg / kg body weight.

28. The method of any one of claims 19 to 24, wherein the GnRH antagonist is administered via a vaginal drug release device.

29. A non-therapeutic method of inducing ovulation in a female mammal, the method comprising: I) a) inducing a FWE according to any one of claims 19 to 28; as well as b) administering an effective amount of an ovulation inducing agent at a fixed time after said administering of said GnRH antagonist; or II) a) inducing a FWE according to any one of claims 19 to 28; b) co-administering an effective amount of progesterone or a progesterone analog with the GnRH antagonist; c) stopping said progesterone or progesterone analog at a fixed time after said administration of said GnRH antagonist and administering an effective amount of prostaglandin (PGF) or a PGF analog; as well as d) administering an effective amount of an ovulation inducing agent at a fixed time after discontinuation of said progesterone or progesterone analog and / or administration of said PGF or PGF analog.

30. A non-therapeutic fixed-time artificial insemination method, the method comprising: a) inducing ovulation according to claim 29; and b) artificially inseminating said mammal at a fixed time after said administering of said ovulation inducing agent; optionally further comprising c) collecting one or more embryos at a fixed time after artificial insemination.

31. A non-therapeutic breeding management method, the method comprising: a) inducing ovulation according to the method of claim 29; and b) mating said mammal at a fixed time after said administering said ovulation inducing agent; optionally further comprising c) collecting one or more embryos at a fixed time after mating.

32. A non-therapeutic fixed-time embryo transfer method, the method comprising: a) inducing FWE according to any one of claims 19 to 28, or inducing ovulation according to the method of claim 29; and b) transferring one or more embryos to said female mammal at a fixed time after said administering of said ovulation inducing agent.

33. A non-therapeutic fixed-time superstimulation or superovulation method, the method comprising: I) a) inducing FWE according to the method of claims 19 to 28; and b) administering to said female mammal an effective amount of FSH or a FSH agonist at a fixed time after said administration of said GnRH antagonist, provided that said FSH or FSH agonist is not co-administered with said GnRH antagonist; or II) a) inducing FWE according to the method of claims 19 to 28; b) co-administering an effective amount of progesterone or a progesterone analogue with the GnRH antagonist; c) administering an effective amount of FSH or a FSH agonist to the female mammal at a fixed time after the administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; and d) discontinuing the progesterone or progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue at a fixed time after the administration of the FSH or FSH agonist.

34. The method according to claim 33, further comprising I) c) administering an effective amount of an ovulation inducing agent at a fixed time after the administration of said FSH or FSH agonist; or II) e) administering an effective amount of an ovulation inducing agent at a fixed time after said stopping of said progesterone or progesterone analog and / or administering said PGF or PGF analog.

35. A non-therapeutic fixed-time oocyte collection method, the method comprising: a) inducing FWE according to any one of claims 19 to 28, or inducing ovulation according to claims 23, 27 or 28; and b) collecting one or more oocytes at a fixed time after administering the GnRH antagonist according to any one of claims 19 to 28, administering the FSH according to claim 33I), administering the PGF according to claim 33II), or administering the ovulation inducing agent according to claim 34.

36. The fixed-time superstimulation or superovulation method according to claim 34, further comprising: I) d) artificially inseminating the female mammal at a fixed time after the administration of the ovulation inducing agent; and e) collecting one or more embryos at a fixed time after said artificial insemination; or II) f) artificially inseminating said female mammal at a fixed time after said administration of said ovulation inducing agent; and g) collecting one or more embryos at a fixed time after said artificial insemination.

37. The fixed-time superstimulation or superovulation method according to claim 35 or 36, further comprising: I) d) mating the female mammal at a fixed time after the administration of the ovulation inducing agent; and e) collecting one or more embryos at a fixed time after breeding; or II) f) mating said female mammal at a fixed time after said administering of said ovulation inducing agent; and g) collecting one or more embryos at a fixed time after said mating.

38. The method of any one of claims 29 to 37, wherein the PGF or PGF analogue comprises PGF2a.

39. The method of any one of claims 29 to 38, wherein the ovulation inducing agent comprises GnRH or a GnRH analog or agonist; LH or an LH analog, agonist, conjugate or recombinant product; or estradiol or an estradiol ester, analog or agonist.

40. The method of any one of claims 19 to 39, wherein the mammal is a livestock animal, optionally the livestock animal is bovine, camelid, equine, porcine, ovine or caprine.

41. The method of any one of claims 19 to 39, wherein the mammal is a wild animal, optionally a bison, elk, reindeer, deer, musk ox, non-human primate, canine or feline.

42. The method of any one of claims 19 to 39, wherein the mammal is a human.

43. The method of any one of claims 29 to 42, wherein the progesterone or progesterone analogue is administered via a vaginal drug release device.

44. The method of claim 43, wherein the mammal is a cow.

45. The method of claim 44, wherein the progesterone or progesterone analog drug releasing device is administered for 7-8 days.

46. ​​The method of claim 45, wherein the GnRH antagonist is administered on day 0 (D0), and the method further comprises i) inserting a progesterone or progesterone analog drug releasing device on D0; ii) administering an effective amount of prostaglandin (PGF2a) or a prostaglandin analog and removing the progesterone or progesterone analog drug releasing device on day 8 (D8); and iii) administering an effective amount of GnRH on day 10 (D10).

47. A drug release device comprising a GnRH antagonist.

48. The drug release device of claim 47, wherein the drug release device provides controlled release of the GnRH antagonist over a period of about one day to about two days.

49. The drug release device of claim 47 or 48, wherein the GnRH antagonist is cetrorelix.

50. The drug release device of any one of claims 47 to 49, further comprising progesterone or a progesterone analog, wherein the drug release device provides controlled release of the progesterone or a progesterone analog over a period of at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, or longer than 10 days.

51. A drug release device according to any one of claims 47 to 50, for use in synchronising follicular wave emergence (FWE) in a population of female mammals, for synchronising ovulation in a population of female mammals, for use in a fixed-time superstimulation or superovulation method, for use in a fixed-time embryo transfer method or for use in a fixed-time artificial insemination method.

52. A non-therapeutic method of synchronizing the occurrence of follicular waves in a population of female mammals, the method comprising inserting a drug release device according to any one of claims 47 to 50 into each mammal in the population of mammals.

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