Use of GnRH antagonists in mammals for synchronizing the appearance of follicular waves

Abstract

The present disclosure relates to methods for mammalian reproductive management using GnRH antagonists such as cetrorelix. Specifically, the GnRH antagonist can be used to synchronize the appearance of follicular waves in a population of female mammals and / or in timed reproductive management protocols such as oocyte recovery protocols, embryo recovery protocols, artificial insemination protocols, or embryo transfer protocols. Also described are devices and kits for reproductive management that include a GnRH antagonist and, optionally, one or more additional drugs useful for reproductive management.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 390,080, filed on July 18, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] Incorporation of Sequence Listing The computer - readable form sequence listing “13764 - P65604PC00_SequenceListing.XML” (4452 bytes), created on July 17, 2023, is hereby incorporated by reference in this specification.

[0003] Field The present disclosure relates generally to methods, uses, devices, and kits for reproductive management in mammals using GnRH antagonists. In particular, the present disclosure relates to the use of GnRH antagonists to synchronize the follicular wave emergence (FWE) in mammals. The methods, uses, devices, and kits are applicable in the synchronization of ovulation, timed artificial insemination protocols, and other reproductive management strategies.

Background Art

[0004] Introduction Existing animal breeding management protocols for the management of donors / recipients for timed artificial insemination and embryo transfer (mainly beef cattle and sheep) incorporate the synchronization of the emergence of new follicular waves as a fundamental reset point. This concept has not been clinically implemented in humans. The methods currently used to synchronize the follicular wave emergence (FWE) in animals include the use of estradiol, gonadotropin-releasing hormone / luteinizing hormone (GnRH / LH), prostaglandin (PGF), or ultrasound-guided ablation of the dominant follicle, or combinations of these various treatments. Estradiol is the most effective drug, and single-injection treatments are widely used in countries in South America, but are prohibited by the European Union for reproductive management in food animals. Estradiol is not favored in other countries (including the USA and Canada) because of environmental concerns around estrogen compounds. Single-treatment with GnRH / LH and / or prostaglandin (either as monotherapy or in combination with a single GnRH / LH injection combined with a single prostaglandin injection) is not effective because it is dependent on the induction of ovulation. Combination protocols, such as GnRH + progesterone controlled internal drug release (CIDR)-PGF-GnRH (e.g., the Obsynch protocol or the Cosynch protocol), and multiple-dose protocols (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. Conservatively estimated, in Brazil alone (one of the largest markets), more than 15 million beef cattle receive artificial insemination annually.

[0005] Ultrasonic examination research in the early 1990s characterized the pattern of follicle growth in beef cattle, and thus the bovine subfamily animal model is widely used to understand the pattern in other mammalian species, including humans. Follicle growth in beef cattle occurs in a wave-like pattern, and most of the estrous cycle includes two to three waves. In beef cattle, each follicular wave is characterized by the growth of small follicles (2-4 mm in diameter) between 8 and 41. Each wave appears after a surge of follicle-stimulating hormone (FSH). At the start of the wave, all follicles require FSH for growth. Therefore, the appearance of follicular waves is temporally related to circulating FSH levels in beef cattle and other mammals. Bovine subfamily animal follicles grow at approximately the same rate for approximately two days, after which one follicle is "selected" to become the dominant follicle, while the remaining follicles stop growing and regress (Ginther et al., 1989; Knopf et al., 1989). The selection of the dominant follicle (about 8 mm in size) is due to feedback from follicular products to the hypothalamus and pituitary gland (Adams and Singh, 2021), and is accompanied by a decrease in circulating FSH levels (Adams et al., 1992). All follicles in the wave have a growth phase (increasing diameter), a static phase (constant diameter), and a regression phase (decreasing diameter). The cells that will become the dominant follicle begin to specifically express luteinizing hormone (LH) receptors at a time close to the time of selection (three days after the appearance of the wave in beef cattle), while subordinate follicles have little or no LH receptors. Plasma FSH levels decrease between the time of wave appearance and the time of dominant follicle selection. Studies have shown that the granulosa cells of the dominant follicle in bovine subfamily animals require LH receptors around the time of selection, and thus the dominant follicle continues to grow by receiving support from LH even at the time when FSH is at its lowest (Adams and Singh, 2021). The dominant follicle produces substances (mainly estradiol) that keep FSH low and prevent the appearance of the next wave. When the dominant follicle regresses or ovulates, the follicular products (e.g., estradiol) decrease to a concentration low enough to allow a surge in circulating FSH concentration, causing a new wave to appear.

[0006] Cattle and camels release one oocyte / oocyte at the time of ovulation (i.e., both are monotocous mammals) and give birth to one offspring. Sheep and goats are polyovulatory species and give birth to 2 to 3 kids / lamb. The mechanisms controlling the appearance of waves and follicular growth are similar among cows, sheep, goats, and camels. However, in contrast to other ruminant species, camels are classified as induced ovulators, and thus, a mating stimulus is required for ovulation to occur (San Martin et al., 1968; England et al., 1969; Fernandez-Baca et al., 1970a). The presence of a functional dominant follicle plays an important role in the ability of camels to accept mating and ovulate (Adams et al., 1990; Bravo et al., 1991; Vaughan et al., 2004). If mating (and subsequent ovulation) does not occur, the development of the dominant follicle continues in a wave-like pattern, which is characterized by an intermittent increase in the number of follicles and selection of the dominant follicle every 20 to 25 days (wave interval), followed by regression (Adams et al., 1990; Vaughan et al., 2004). To simplify flock management methods, ovulation in llama herds can be synchronized for artificial insemination by inducing new follicular waves. Hormonal studies conducted in camels have attempted to induce ovulation using 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), but the results are still varied.

[0007] The selection of dominant follicles and ovulation both require hormonal stimulation from FSH and LH (Adams et al., 2008). Both FSH and LH are released from the anterior pituitary gland following the secretion of GnRH from the hypothalamus. In human assisted reproduction, the endogenous GnRH membrane receptors (present in the pituitary gland) are generally inhibited by GnRH antagonists such as cetrorelix (Kovacs et al., 2001). Cetrorelix (trade name: Cetrotide®) is used as a fertility treatment for women to avoid premature increases in LH during ovarian stimulation (EMD Serono Inc., 2008). The ability of cetrorelix to prevent ovulation of existing follicles is well known and thus it is clinically used in human FSH stimulation protocols for assisted reproduction. In such protocols, cetrorelix is administered simultaneously with FSH to block ovulation of FSH-induced follicles.

[0008] Cetrorelix is a 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) that acts as a GnRH / LHRH antagonist. Cetrorelix occupies the GnRH receptors on gonadotropin-secreting cells in the anterior pituitary gland, thereby preventing the release of LH even in the presence of GnRH pulses. In human assisted reproduction protocols, cetrorelix is used as a once-daily subcutaneous (s / c) injection of 0.25 mg for up to 6 days or as an initial s / c bolus of 3 mg after the first 4 days followed by a once-daily administration of 0.25 mg. Its elimination half-life is quite short (5 hours after a single injection of 0.25 mg in women).

[0009] Cetrorelix is known to decrease circulating LH concentrations, inhibit the growth of existing single dominant follicles or multiple large follicles (e.g., during ovarian FSH stimulation), and prevent premature ovulation. Cetrorelix is clinically used in reproductive assistance technologies for women (e.g., to prevent premature ovulation during FSH stimulation in human IVF cycles). Other GnRH antagonists are also used for this purpose in reproductive assistance in humans. During human FSH stimulation protocols, since multiple follicles grow to ovulatory size, cetrorelix is co-administered with FSH to prevent ovulation of existing multiple dominant follicles at the time of treatment.

[0010] The effects of GnRH antagonists such as cetrorelix have been studied in beef cattle and other farm animals.

[0011] Nivet et al. (2018) investigated the mechanisms underlying the oocyte competence when LH was inhibited by cetrorelix. In heifers, at the end of superovulation, cetrorelix treatment was performed for 4 days. A significant reduction in the number of follicles >10 mm in diameter was observed during the developmental stage of the dominant follicle.

[0012] Haughian et al. (2013) described the effect of asrelin, a GnRH antagonist, on follicular development. Asrelin was administered immediately before ovulation, and multiple injections were continued after ovulation and the appearance of follicular waves. Asrelin did not reduce the concentration of FSH during the preovulatory surge, and early follicular development was not affected, but the subsequent growth of the dominant follicles (>8.0 mm) was inhibited by asrelin. The lack of selection of dominant follicles in the asrelin group was associated with a decrease in LH concentration, but not with FSH. The maximum diameter of the dominant follicle in control animals (13.3 mm) was larger than that in animals treated with asrelin (7.7 mm).

[0013] Ginther et al. (2012) described the effect of aserine on follicular development. Aserine was administered to heifers on days 15, 16, and 17 after ovulation. Aserine regressed the dominant follicle of the second wave and induced the third wave. Heifers treated with aserine had an ovulatory interval of 25 to 34 days, whereas untreated heifers had an interval of 19 to 23 days. Synchronization of wave emergence was not examined or discussed.

[0014] Silva et al. (2011) described the effect of cetrorelix on follicular development in llamas. Cetrorelix inhibited the preovulatory LH surge and ovulation induced by GnRH and ovulation-inhibiting factor (OIF) in llamas.

[0015] Ulker et al. (2001) reported that cetrorelix suppresses the secretion of LH and progesterone in beef cattle without affecting the length of the estrous cycle.

[0016] Oussaid et al. (2000) described the effect of antarelix, a GnRH antagonist, on follicular development. Treatment of superstimulated heifers with antarelix for 36 - 60 hours resulted in follicular atresia without affecting the competence of oocytes as determined by in vitro embryo production.

[0017] Fike et al. (1997) described the effect of SB - 75, an LHRH antagonist, on follicular development. Treatment of animals with SB - 75 from day 2 to day 12 of the estrous cycle changed circulating LH and FSH, suppressed the survival of the dominant follicle, and secreted estradiol. The effect of SB - 75 on the size and number of follicles of the dominant follicle was described.

[0018] Madill et al. (1994) described the effects of SB-75 on hormone levels and ovulation, and SB-75 can be used to delay the LH surge (detecting and analyzing blood samples by radioimmunoassay) and ovulation (detecting by ultrasound) in superovulated heifers.

[0019] Leonardi (2018) experimentally utilized GnRH antagonists in beef cattle to test the effects of kisspeptin. This study involved the administration of cetrorelix on day 6 of follicular development with a low plasma progesterone concentration.

Prior Art Documents

Patent Documents

[0020]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0021] There is a need to develop a novel method for manipulating follicular waves in large and small ruminants, camelids, and other livestock that is effective and efficient when implemented under field conditions. As described herein, the inventors have demonstrated that the administration of cetrorelix, a GnRH antagonist, elicits the emergence of new follicular waves within a consistent time frame in female mammals such as beef cattle and alpacas. Accordingly, the present disclosure relates to the use of GnRH antagonists for the reproductive management of female mammals. The methods and uses described herein are not therapeutic.

Means for Solving the Problems

[0022] One aspect includes a non-therapeutic method for synchronizing the emergence of follicular waves (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.

[0023] In one embodiment, the GnRH antagonist includes cetrorelix, asirelin, antarelix / teverelix, degarelix, ganirelix, antide, leuprorelin, elagolix, abarelix, plazarelix, ramorelix, antide, detirelix, ocrelizumab, linzagolix, opigolix, spugolix, and A-75998. In one embodiment, the GnRH antagonist includes abarelix. In one embodiment, the GnRH antagonist includes degarelix. In one embodiment, the GnRH antagonist includes leuprorelin. In one embodiment, the GnRH antagonist includes cetrorelix.

[0024] In one embodiment, the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

[0025] In one embodiment, the administration of the GnRH antagonist includes at least one treatment, and optionally two or three treatments.

[0026] In one embodiment, the GnRH antagonist includes cetrorelix delivered by intramuscular injection at a dose of about 2.5 μg per kg of body weight to about 40 μg per kg of body weight, and optionally at a dose of about 5 μg / kg, 7.5 μg / kg, 10 μg / kg, or 20 μg per kg of body weight.

[0027] In one embodiment, the GnRH antagonist is administered by a vaginal drug delivery device.

[0028] In one embodiment, the method further includes co-administering progesterone or a progesterone analog, and optionally, the progesterone or progesterone analog is administered by a vaginal drug delivery device.

[0029] In one embodiment, the mammal is a breeding livestock, and optionally, the breeding livestock is a species of Bovinae, Equidae, Equus, Sus, Ovis, or Capra.

[0030] In one embodiment, the mammal is a pet and a wild animal, and in some cases, it is a bison, a pronghorn, a caribou, a deer, a yak, a non-human primate, a dog, or a cat, and in some cases, it is a tiger, a lion, or a leopard.

[0031] In one embodiment, the mammal is a human.

[0032] In one embodiment, the population is a mixed population.

[0033] In one embodiment, the method comprises, at regular intervals after administration of a GnRH antagonist, inducing ovulation in each mammal of a population of female mammals, I) administering an effective amount of an ovulation inducer at regular intervals after administration of the GnRH antagonist, or II) co-administering an effective amount of progesterone or a progesterone analog with the GnRH antagonist; discontinuing progesterone or a progesterone analog at regular intervals after administration of the GnRH antagonist, and administering an effective amount of prostaglandin (PGF) or a PGF analog; and administering an effective amount of an ovulation inducer at regular intervals after discontinuation of progesterone or a progesterone analog and / or after administration of PGF or a PGF analog further comprising the step of inducing ovulation.

[0034] In one embodiment, the method further comprises, at regular intervals after administration of the ovulation inducer, artificially inseminating each mammal of the population of female mammals.

[0035] In one embodiment, the method further comprises, at regular intervals after administration of the ovulation inducer, mating each mammal of the population of female mammals.

[0036] In one embodiment, the method further comprises, at regular intervals after administration of the ovulation inducer, transplanting an embryo into each animal of the population of female mammals.

[0037] Another aspect is a non-therapeutic method for synchronizing ovulation in a population of female mammals, I) a) Administering an effective amount of a GnRH antagonist; and b) After administration of the GnRH antagonist, periodically administering an effective amount of an ovulation inducer to each mammal in a population of female mammals; or II) a) Co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue; b) After administration of the GnRH antagonist, periodically discontinuing the progesterone or progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue; and c) After discontinuation of the progesterone or progesterone analogue and / or administration of the PGF or PGF analogue, periodically administering an effective amount of an ovulation inducer to each mammal in a population of female mammals comprising a method.

[0038] Another aspect is a non-therapeutic method of inducing the appearance of follicular waves in a female mammal, comprising administering an effective amount of a GnRH antagonist to the female mammal.

[0039] Another aspect is a non-therapeutic method of inducing ovulation in a female mammal, I) a) Administering an effective amount of a GnRH antagonist to the female mammal; and b) After administration of the GnRH antagonist, periodically administering an effective amount of an ovulation inducer; or II) a) Co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue to the female mammal; b) After administration of the GnRH antagonist, periodically discontinuing the progesterone or progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue; and c) After discontinuation of the progesterone or progesterone analogue and / or administration of the PGF or PGF analogue, periodically administering an effective amount of an ovulation inducer comprising a method.

[0040] Another aspect is a non-therapeutic timed embryo transfer method, I) a) Administering an effective amount of a GnRH antagonist to a female mammal; b) At regular intervals after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer; and c) At regular intervals after administration of the ovulation inducer, transplanting one or more embryos into the female mammal, or II) a) Simultaneously administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue to a female mammal; b) At regular intervals after administration of the GnRH antagonist, discontinuing the progesterone or progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue; c) At regular intervals after discontinuing the progesterone or progesterone analogue and / or administering the PGF or PGF analogue, administering an effective amount of an ovulation inducer; and d) At regular intervals after administration of the ovulation inducer, transplanting one or more embryos into the female mammal comprising a method.

[0041] Another aspect is a non-therapeutic timed superovulation method or superovulation method, I) a) Administering an effective amount of a GnRH antagonist to a female mammal; b) Under the condition that FSH or an FSH agonist is not co-administered with the GnRH antagonist, at regular intervals after administration of the GnRH antagonist, administering an effective amount of FSH or an FSH agonist to the female mammal; and c) At regular intervals after administration of the FSH or FSH analogue, administering an effective amount of an ovulation inducer, or II) a) Simultaneously administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue to a female mammal; b) Under the condition that FSH or an FSH agonist is not co-administered with the GnRH antagonist, at regular intervals after administration of the GnRH antagonist, administering an effective amount of FSH or an FSH agonist to the female mammal; c) At regular intervals after administration of the FSH or FSH agonist, discontinuing the progesterone or progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue; and d) At regular intervals after discontinuing the progesterone or progesterone analogue and / or administering the PGF or PGF analogue, administering an effective amount of an ovulation inducer including a method.

[0042] In one embodiment, the method further includes the step of collecting one or more oocytes at regular intervals after administration of an ovulation inducer. In one embodiment, the method further includes the steps of: I) d) or II) e) artificially inseminating a female mammal at regular intervals after administration of an ovulation inducer, and I) e) or II) f) collecting one or more embryos at regular intervals after artificial insemination. In one embodiment, the method further includes the steps of: I) d or II) e) mating a female mammal at regular intervals after administration of an ovulation inducer, and I) e) or II) f) collecting one or more embryos at regular intervals after artificial insemination.

[0043] Another aspect is a non-therapeutic timed embryo collection method, comprising 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 the female mammal at regular intervals after administration of the GnRH antagonist, provided that FSH or an FSH agonist is not co-administered with the GnRH antagonist; c) administering an effective amount of an ovulation inducer at regular intervals after administration of FSH or an FSH analog; d) artificially inseminating or mating the female mammal at regular intervals after administration of the ovulation inducer; and e) collecting one or more embryos at regular intervals after artificial insemination or mating, or II) a) Administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue to a female mammal simultaneously; b) Administering an effective amount of FSH or an FSH agonist to the female mammal at regular intervals after administration of the GnRH antagonist, provided that the FSH or FSH agonist is not administered simultaneously with the GnRH antagonist; c) Discontinuing progesterone or a progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue at regular intervals after administration of the FSH or FSH agonist; d) Administering an effective amount of an ovulation inducer at regular intervals after discontinuation of progesterone or a progesterone analogue and / or after administration of PGF or a PGF analogue; e) Artificially inseminating or mating the female mammal at regular intervals after administration of the ovulation inducer; and f) Recovering one or more embryos at regular intervals after artificial insemination or mating comprising a method.

[0044] Another aspect is a non-therapeutic method for timed oocyte retrieval, comprising I) a) Administering an effective amount of a GnRH antagonist; and b) Recovering one or more oocytes at regular intervals after administration of the GnRH antagonist II) 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 the female mammal at regular intervals after administration of the GnRH antagonist, provided that the FSH or FSH agonist is not administered simultaneously with the GnRH antagonist; and c) Recovering one or more oocytes at regular intervals after administration of the FSH or FSH agonist III) 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 the female mammal at regular intervals after administration of the GnRH antagonist, provided that the FSH or FSH agonist is not administered simultaneously with the GnRH antagonist; c) Administering an effective amount of an ovulation inducer at regular intervals after administration of the FSH or FSH analogue; and d) Recovering one or more oocytes at regular intervals after administration of the ovulation inducer, or IV) a) Administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal simultaneously; b) Administering an effective amount of FSH or an FSH agonist to the female mammal at regular intervals after administration of the GnRH antagonist, provided that the FSH or FSH agonist is not co-administered with the GnRH antagonist; c) Discontinuing progesterone or a progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog at regular intervals after administration of the FSH or FSH agonist; d) Administering an effective amount of an ovulation inducer at regular intervals after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog; and e) Recovering one or more oocytes at regular intervals after administration of the ovulation inducer comprising a method.

[0045] Another aspect is a non-therapeutic timed artificial insemination method, I) a) Administering an effective amount of a GnRH antagonist to a female mammal; b) Administering an effective amount of an ovulation inducer to the female mammal at regular intervals after administration of the GnRH antagonist; and c) Artificially inseminating the female mammal at regular intervals after administration of the ovulation inducer, or II) a) Administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal simultaneously; b) Discontinuing progesterone or a progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog at regular intervals after administration of the GnRH antagonist; c) Administering an effective amount of an ovulation inducer at regular intervals after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog; and d) Artificially inseminating the female mammal at regular intervals after administration of the ovulation inducer comprising a method.

[0046] Another aspect is a breeding management method, I) a) Administering an effective amount of a GnRH antagonist to a female mammal; b) At regular intervals after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer; and c) At regular intervals after administration of the ovulation inducer, mating the female mammal, or II) a) Simultaneously administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog to a female mammal; b) At regular intervals after administration of the GnRH antagonist, discontinuing the progesterone or progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog; c) At regular intervals after discontinuation of the progesterone or progesterone analog and / or administration of the PGF or PGF analog, administering an effective amount of an ovulation inducer; and d) At regular intervals after administration of the ovulation inducer, mating the female mammal comprises a method comprising

[0047] In one embodiment of the aspects described herein, the GnRH antagonist comprises cetrorelix, abarelix, degarelix, ganirelix, antarelix / teverelix, plazarelix, lamorelix, antide, detirelix, ozelix, asiline, 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.

[0048] In one embodiment of the aspects described herein, the GnRH antagonist is administered by intramuscular injection or subcutaneous injection.

[0049] In one embodiment of the aspects described herein, the administration of the GnRH antagonist comprises at least one treatment, and in some cases two or three treatments.

[0050] In one embodiment of the aspects described herein, the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of from about 5 μg per kg of body weight to about 40 μg per kg of body weight, optionally at a dose of about 20 μg per kg of body weight.

[0051] In one embodiment of the aspects described herein, the GnRH antagonist is administered by a vaginal drug delivery device.

[0052] In one embodiment of the aspects described herein, the mammal is a farm animal, and optionally, the farm animal is a bovine, camelid, equine, porcine, ovine, or caprine. In one embodiment, the mammal is a bovine.

[0053] In one embodiment of the aspects described herein, the mammal is a wild animal, optionally a bison, wapiti, caribou, deer, yak, non-human primate, canine, or feline, optionally a tiger, lion, or leopard.

[0054] In one embodiment of the aspects described herein, the mammal is a human.

[0055] In one embodiment of the aspects described herein, the method further comprises the step of co-administering progesterone, and optionally, the progesterone is administered by a vaginal drug delivery device. In one embodiment, the release of the progesterone drug is carried out for 7 - 8 days.

[0056] In one embodiment of the aspects described herein, the GnRH antagonist is administered on day 0 (D0), and the method further comprises the steps of inserting a progesterone drug release device on D0, administering an effective amount of PGF or a PGF analogue on day 8 (D8), removing the progesterone drug release device, and administering an effective amount of GnRH on day 10 (D10).

[0057] In one embodiment of the aspects described herein, the PGF or PGF analog includes prostaglandin 2 alpha (PGF2a), cloprostenol, dinoprost, dinoprost tromethamine, bimatoprost, travoprost, carboprost, and latanoprost. In one embodiment, the PGF or PGF analog includes PGF2a.

[0058] In one embodiment of the aspects described herein, the ovulation inducer includes GnRH, LH, or estradiol, or an agonist, analog, ester, conjugate, or recombinant product of any of these. In one embodiment, the GnRH or GnRH analog or agonist includes GnRH, buserelin, deslorelin, fertirelin, gonadorelin, goserelin, leuprolerein, or triptorelin. In one embodiment, the LH or LH analog, agonist, conjugate, or recombinant product includes LH, human chorionic gonadotropin / hCG, or equine chorionic gonadotropin / eCG. In one embodiment, the estradiol or estradiol ester, analog, or agonist includes estradiol-17 beta, estradiol benzoate, estradiol valerate, or estradiol cypionate.

[0059] Another aspect includes a drug delivery device including a GnRH antagonist. In one embodiment, the GnRH antagonist includes cetrorelix, abarelix, degarelix, ganirelix, antarelix / teverelix, plazarelix, lamorelix, antide, detirelix, ocrelix, asilin, elagolix, linzagolix, relugolix, opigolix, sufgolix, or A-75998. In one embodiment, the GnRH antagonist includes abarelix. In one embodiment, the GnRH antagonist includes degarelix. In one embodiment, the GnRH antagonist includes relugolix. In one embodiment, the GnRH antagonist is cetrorelix.

[0060] In one embodiment, the drug delivery device provides controlled release of a GnRH antagonist over a period of about 1 day to about 2 days.

[0061] In one embodiment, the drug delivery device further comprises progesterone, and the drug delivery device provides controlled release of 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 a period longer than 10 days.

[0062] In one embodiment, the drug delivery device is for use in synchronizing the appearance of follicular waves (FWE) in a female mammalian population, for use in synchronizing ovulation in a female mammalian population, for use in timed superovulation methods or superovulation methods, for use in timed embryo transfer methods, for use in timed artificial insemination methods, or for use in timed breeding methods.

[0063] One aspect includes a non-therapeutic method of synchronizing the appearance of follicular waves in a female mammalian population, the method including inserting a drug delivery device containing a GnRH antagonist as described herein into each mammal of the mammalian population.

[0064] The foregoing sections are provided by way of example only and are not intended to limit the scope of the present disclosure and the appended claims. Additional objects and advantages related to the compositions and methods of the present disclosure will be understood by those skilled in the art in light of the current claims, specification, and examples. For example, the various aspects and embodiments of the present disclosure can be utilized in many combinations, all of which are expressly contemplated herein. These additional advantages, objects, and embodiments are expressly included within the scope of the present disclosure. Publications and other materials used herein to explain the background of the present disclosure and to provide additional details regarding its implementation in specific cases are incorporated by reference and are listed in the appended references section for convenience.

[0065] Further objects, features, and advantages of the present disclosure will become apparent from the following detailed description in combination with the accompanying drawings that illustrate exemplary embodiments of the present disclosure.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0067] The following is a detailed description provided to assist those skilled in the art in the implementation of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the present disclosure. All publications, patent applications, patents, drawings, and other references mentioned herein are hereby incorporated by reference in their entirety.

[0068] Furthermore, the definitions and embodiments described in a particular section are applicable to other embodiments herein that would be understood by those skilled in the art to be suitable. For example, in a following section, different aspects of the present disclosure are defined in more detail. Each aspect so defined can be combined with any one or more other aspects, unless it is clearly indicated otherwise. In particular, any feature described herein can be combined with any one or more other features described herein.

[0069] The present disclosure relates to the non-therapeutic use of GnRH antagonists for the reproductive management of mammals. As described herein, the inventors have demonstrated that administration of cetrorelix, a GnRH antagonist, synchronizes the appearance of new follicular waves in female mammals such as beef cattle and alpacas. For example, administration of a single 1.5 mg injection of cetrorelix in an unsynchronized alpaca population resulted in the appearance of a new follicular wave after an average of 5.33 days (dispersion of 7.38), whereas in control animals it was an average of 8.93 days (dispersion of 22.067). The smaller dispersion seen in the cetrorelix treatment group (7.38 vs. 22.067) indicates that the appearance of the FWE was more synchronized than in the control. Since the mechanisms controlling follicular dynamics are similar in all mammals, this synchronized FWE after administration of a GnRH antagonist such as cetrorelix is expected to be reproduced in other mammalian species, with species-specific temporal dynamics. Accordingly, cetrorelix (and related GnRH receptor antagonists) can be used to manipulate the appearance of the next follicular wave in female mammals, including, for example, beef cattle, other farm animals, wild mammals, non-human primates, and in humans. The methods and uses described herein are non-therapeutic.

[0070] The use of cetrorelix has been found to prevent premature ovulation of multiple leading follicles in humans during FSH-stimulated IVF cycles. Conversely, the approach described herein enables the use of a single injection or vaginal application of a GnRH antagonist (e.g., in combination with progesterone in some cases as part of a vaginal drug delivery device) at any stage of the ovarian cycle to reset the emergence of the next (new) follicular wave at a consistent time point from the time of treatment. Without wishing to be bound by theory, the synchronized emergence of follicular waves with cetrorelix is thought to rely on the fact that LH consistently declines simultaneously in all animals. This may involve the same endocrine pathway as the action of estradiol on the emergence of waves, but at different stages (estradiol prevents GnRH release, while cetrorelix blocks LH release). This "synchronization of follicular waves" is the basis for synchronization of ovulation and timed artificial insemination protocols in beef cattle and other farm animals, as well as gonadotropin stimulation and superovulation treatments for embryo production / transfer.

[0071] A single injection of cetrorelix or other GnRH antagonist compounds is an alternative to the use of estradiol and / or GnRH for synchronization of follicular waves in mammals (including humans) because it is non-steroidal, easy and inexpensive to synthesize, effective at all stages of the leading follicle, and only a single treatment is required. Furthermore, cetrorelix or other GnRH antagonists can be impregnated into silicone or similar compounds as an immediate release drug for preparing a vaginal drug delivery device to simplify the treatment (a single device that does not require injection) and prevent a second use of previously used devices, in some cases in combination with progesterone.

[0072] I. Definitions As used herein, the following terms may have the meanings given to them below, unless specifically stated otherwise. However, it should be understood that other meanings, which are known or understood by those skilled in the art and which are within the scope of the present disclosure, are also possible. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the definition including this specification shall prevail. Further, the materials, methods, and examples are illustrative only and not limiting.

[0073] Where a range of values is indicated, each value between the upper and lower limits of the range, to one-tenth of the unit of the lower limit, unless otherwise clearly indicated from the context, and every other described value or value that exists between the described range, is included herein. Any smaller range from any lower limit to any upper limit is considered. The upper and lower limits of such smaller ranges, which can be independently included in these smaller ranges, are also included herein, to the extent that there are any specifically excluded limits in the described range. Where the described range includes one or both of the limits, ranges excluding either or both of these included limits are also included herein.

[0074] All numerical values in the detailed description and claims of this specification are modified by the values indicated by "about" or "approximately", taking into account experimental errors and variations expected by those skilled in the art. For example, "about" can mean plus or minus 10% of the indicated value being referred to, or plus or minus 5%.

[0075] As used herein, the singular forms "a", "an", and "the" include references to the plural, unless the context clearly indicates otherwise.

[0076] As used herein, the expression "and / or" shall be understood to mean "either or both" of the elements so combined, i.e., elements that in some cases coexist and in other cases do not coexist. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so combined. Other elements, whether or not specifically identified in relation to a specifically identified element, may in some cases exist outside of the elements specifically identified by the "and / or" clause.

[0077] As used herein, "or" shall 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 construed as inclusive, i.e., including at least one of many elements or list of elements, but also including more than one, and in some cases also including additional unlisted items. Only terms clearly indicating otherwise, such as "only one" or "exactly one", or "consisting of" when used in the claims, refer to exactly one of many elements or list of elements. Ordinarily, the term "or" as used herein should only be construed to indicate an exclusive alternative (i.e., "either one or the other, but not both") when preceded by exclusive terms such as "any", "one", "only one", or "exactly one".

[0078] As used herein, all transitional phrases, such as "comprising", "including", "carrying", "having", "containing", "accompanying", "holding", "being composed of", etc., are open-ended, i.e., are understood to mean including without being limited thereto. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively.

[0079] As used herein, the expression "at least one" with reference to an enumeration of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the enumeration of elements, but does not necessarily include at least one of each and every element specifically recited in the enumeration of elements, nor does it exclude any combination of elements in the enumeration of elements. This definition also allows for the possibility that elements other than those specifically identified in the enumeration of elements referred to by the expression "at least one" may, in some cases, exist, whether or not they are related to the specifically identified elements.

[0080] In a particular method described herein that includes two or more steps or acts, it should also be understood that the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless the context indicates otherwise.

[0081] II. Methods and Uses As shown herein, administration of cetrorelix, a GnRH antagonist, at various stages of the ovarian cycle resulted in the emergence of a new follicular wave at a consistent time point from the time of administration. Thus, GnRH antagonists, such as cetrorelix, can be used to synchronize the emergence of new follicular waves and, in some cases, subsequent ovulation in mammalian populations, such as herds of breeding livestock. This synchronized FWE is useful in reproductive management, for example, in timed oocyte recovery protocols, artificial insemination protocols, breeding, and / or induction of synchronized ovulation for embryo production / transfer in breeding livestock. Accordingly, one aspect of the present disclosure is a non-therapeutic method of synchronizing follicular wave emergence (FWE) in a female mammalian population, the method comprising administering an effective amount of a GnRH antagonist to each mammal in the female mammalian population. Another aspect of the present disclosure is a non-therapeutic method of synchronizing ovulation in a female mammalian population, the method comprising administering an effective amount of a GnRH antagonist to each mammal in the female mammalian population and, at a timed interval after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer. As is understood in the art, in some cases, for example, in species that are natural ovulators (e.g., bovines, sheep, horses, pigs, dogs), individual mammals within the population may have existing corpora lutea that can interfere with synchronization. Thus, additional treatments can be performed to avoid the need to examine individual animals for the presence of existing functional corpora lutea. For example, progesterone can be co-administered with the GnRH antagonist a) to prevent the LH surge and ovulation, b) to prime the endometrium to prevent early PGF release after ovulation, and c) because withdrawal of progesterone causes an ovulatory cascade. PGF can be administered around the time of progesterone withdrawal to induce regression of all existing corpora lutea.Accordingly, in one aspect, a non-therapeutic method for synchronizing ovulation in a mammalian population comprises the steps of administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog; at a timed interval after administration of the GnRH antagonist, discontinuing administration of the progesterone or progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog; and at a timed interval after discontinuation of the progesterone or progesterone analog and / or administration of the PGF or PGF analog, administering an effective amount of an ovulation inducer to each mammal in the female mammalian population. The methods and uses described herein are non-therapeutic.

[0082] The GnRH antagonist can be administered at any stage of the ovulatory cycle. Thus, in various aspects and embodiments of the methods described herein, it is not necessary to know the stage of the cycle of the female mammal and / or the population can be a mixed population.

[0083] In various embodiments of the aspects described herein, the method further comprises the step of recovering one or more oocytes from each mammal, the step of artificially inseminating each mammal, the step of mating each mammal, the step of recovering one or more embryos from each mammal, or the step of transplanting embryos to each animal in the female mammalian population at a timed interval after administration of the ovulation inducer.

[0084] Another aspect described herein is a non-therapeutic method of inducing the appearance of follicular waves in female mammals, the method comprising administering to the female mammal an effective amount of a GnRH antagonist. A further aspect is a non-therapeutic method of inducing ovulation in a female mammal, comprising: I) a) administering to the female mammal an effective amount of a GnRH antagonist; and b) at a timed interval after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer; or II) a) co-administering to the female mammal an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog; b) at a timed interval after administration of the GnRH antagonist, discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog; and c) at a timed interval after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog, administering an effective amount of an ovulation inducer.

[0085] Another aspect of the present disclosure is a non-therapeutic timed artificial insemination method, comprising: I) a) administering to the female mammal an effective amount of a GnRH antagonist; b) at a timed interval after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer; and c) at a timed interval after administration of the ovulation inducer, artificially inseminating the female mammal; or II) a) co-administering to the female mammal an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog; b) at a timed interval after administration of the GnRH antagonist, discontinuing the administration of progesterone or a progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog; c) at a timed interval after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog, administering an effective amount of an ovulation inducer; and d) at a timed interval after administration of the ovulation inducer, artificially inseminating the female mammal.

[0086] Another aspect of the present disclosure is 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 inducer at regular intervals after the administration of the GnRH antagonist; and c) mating the female mammal after the administration of the ovulation inducer; or II) a) simultaneously 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 at regular intervals after the administration of the GnRH antagonist and administering an effective amount of prostaglandin (PGF) or a PGF analog; c) administering an effective amount of an ovulation inducer at regular intervals after the discontinuation of progesterone or a progesterone analog and / or after the administration of PGF or a PGF analog; and d) mating the female mammal after the administration of the ovulation inducer. In these various aspects, it is not necessary to know the stage of the cycle of the female mammal, and / or the method can be used in a mixed population of female mammals.

[0087] Another aspect of the present disclosure is a gonadotropin stimulation and superovulation method, optionally for oocyte retrieval 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 regular intervals after the administration of the GnRH antagonist; or II) a) co-administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analogue to a female mammal; b) administering an effective amount of FSH at regular intervals after the administration of the GnRH antagonist; and c) discontinuing the administration of progesterone or a progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue at regular intervals after the administration of FSH. In one embodiment, the method further comprises: I) c) administering an effective amount of an ovulation inducer at regular intervals after the administration of FSH; or II) d) administering an effective amount of an ovulation inducer at regular intervals after the discontinuation of progesterone or a progesterone analogue and / or after the administration of PGF or a PGF analogue. In one embodiment, for example, for in vitro fertilization / embryo production, multiple oocytes are retrieved after the administration of a GnRH antagonist, FSH, PGF or a PGF analogue, or an ovulation inducer. In one embodiment, after the administration of a GnRH antagonist, FSH, PGF or a PGF analogue, or an ovulation inducer, artificial insemination of the animal is performed, and optionally, multiple embryos are retrieved approximately 5 to 8 days after artificial insemination. In these various aspects and embodiments, it is not necessary to know the stage of the female mammal's cycle, and / or the method can be used in a mixed population of female mammals.

[0088] Also provided herein is the use of GnRH antagonists to synchronize the appearance of follicular waves and / or ovulation in a female mammalian population. Further provided herein is the use of GnRH antagonists to induce the appearance of follicular waves in female mammals. Further provided herein is the use of GnRH antagonists to induce ovulation in female mammals. Further provided herein is the use of GnRH antagonists in timed artificial insemination methods. Further provided herein is the use of GnRH antagonists, optionally in gonadotropin stimulation and superovulation procedures, for embryo production / transfer methods. In these embodiments, it is not necessary to know the stage of the female mammalian cycle and / or the use may be in a mixed population of female mammals.

[0089] Also disclosed herein is the use of GnRH antagonists, optionally cetrorelix, in the manufacture of a medicament for synchronizing the appearance of follicular waves in a female mammalian population. One embodiment includes the use of GnRH antagonists, optionally cetrorelix, in the manufacture of a medicament for synchronizing ovulation in a female mammalian population. One embodiment includes the use of GnRH antagonists in the manufacture of a medicament for inducing the appearance of follicular waves in female mammals. One embodiment includes the use of GnRH antagonists in the manufacture of a medicament for inducing ovulation in female mammals. One embodiment includes the use of GnRH antagonists in the manufacture of a medicament for timed artificial insemination methods and / or for gonadotropin stimulation and superovulation procedures for embryo production / transfer. In these embodiments, it is not necessary to know the stage of the female mammalian cycle and / or the medicament may be for use in a mixed population of female mammals.

[0090] In a further aspect of the disclosure, a GnRH antagonist, in some cases cetrorelix, is for use in synchronizing the appearance of follicular waves in a female mammalian population. In one aspect, the GnRH antagonist is for use in synchronizing ovulation in a female mammalian population. In one aspect, the GnRH antagonist is for use in inducing the appearance of follicular waves in a female mammal. In one aspect, the GnRH antagonist is for use in inducing ovulation in a female mammal. In one aspect, the GnRH antagonist is for use in a timed artificial insemination method. In another aspect, the GnRH antagonist is for use in gonadotropin stimulation and superovulation procedures for embryo production / transfer. In these aspects, it is not necessary to know the stage of the female mammalian cycle and / or the GnRH antagonist can be for use in a mixed population of female mammals.

[0091] As used herein, the term "GnRH antagonist" refers to an agent that reduces, decreases, or otherwise blocks the activity of GnRH and / or the GnRH receptor, including but not limited to small molecules, peptides, and antibodies (and fragments thereof). GnRH antagonists are also described in the literature as luteinizing hormone-releasing hormone (LHRH) antagonists. Commonly used GnRH antagonists include, but are not limited to, the peptides cetrorelix, abarelix, degarelix, ganirelix, antarelix / teverelix, plazarelix, lamorelix, antide, detirelix, ozelix, asrelin, and the small molecule compounds elagolix, linzagolix, relugolix, opigolix, sufgolix, and A-75998 (see also Table 24). Accordingly, in one embodiment, the GnRH antagonist is cetrorelix, abarelix, degarelix, ganirelix, antarelix / teverelix, plazarelix, lamorelix, antide, detirelix, ozelix, asrelin, 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.

[0092] As used herein, "cetrorelix" (trade name: Cetrotide™) 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-alanine amide.

[0093] As used herein, "abarelix" (Plenaxis™) 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-asparagyl-L-leucyl-N6-isopropyl-L-lysyl-L-prolyl-D-alanine amide.

[0094] As used herein, "degarelix" (trade name: Firmagon™) refers to N-acetyl-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridyl)-D-alanyl-L-seryl-4-((S)-dihydroorotamide)-L-phenylalanyl-4-ureido-D-phenylalanyl-L-leucyl-N6-isopropyl-L-lysyl-L-prolyl-D-alanine amide.

[0095] As used herein, "relugolix" (trade names: Orgovyx™, Relumina™) 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.

[0096] The term "follicular wave" refers to the stage in the ovarian cycle that corresponds to the simultaneous growth of multiple follicles, followed by the selection and continued growth of one or more (species-dependent) dominant follicles and the regression of the remaining subordinate follicles. Thus, the terms "appearance of the follicular wave", "FWE", "wave appearance", and variants thereof refer to the beginning or initial stage of the follicular wave. In beef cattle and alpacas, FWE can be retrospectively defined as the first detection of a dominant follicle of 4 or 5 mm size, accompanied by an increase in the number of follicles of 3 - 4 mm.

[0097] The expressions "synchronization of the appearance of the follicular wave", "synchronize the appearance of the follicular wave", and variants thereof, when used with respect to a population of female mammals in different (i.e., random) stages of the ovarian cycle, mean that in individual animals, the appearance of the follicular wave occurs at approximately the same time or within the same time span as the remaining animals in the population. For example, FWE can occur in individual animals on the same day as, or within one or two days of, other animals in the population. As will be understood by those skilled in the art, synchronized FWE in an animal population enables synchronization of the ovulation induction method within the population, thereby resulting in synchronized ovulation in the animal population.

[0098] The timing of FWE after administration of a GnRH antagonist varies depending on various factors such as the drug or compound administered, the dose administered, the pharmaceutical formulation, the route of administration, the mammalian species being treated, etc. The timing of FWE for a given combination of factors (e.g., species, drug, dose, etc.) can be determined experimentally using methods known in the art, for example, using ultrasound (e.g., repeated ultrasound examinations at 12 to 24 - hour intervals).

[0099] As used herein, the term "fixed time" means a specific or predetermined amount of time between one event, such as a step or process in a method, and another event, such as another step or another process in the method. As will be understood by those skilled in the art, the timing ("fixed time") of the next event or process in the methods described herein depends on the various factors shown above (e.g., species, drug, dosage, etc.). For example, a new FWE occurs at a fixed time after administration of a GnRH antagonist. In the examples shown herein, in heifers, a new FWE is observed at a fixed time approximately 3.40 ± 0.75 days after administration of a single intramuscular dose of cetrorelix acetate at a dosage of 20 μg per kg of body weight, approximately 3.5 ± 0.4 days after administration of a single intramuscular dose of cetrorelix at a dosage of 3 mg (approximately 7.5 μg per kg of body weight), or approximately 5.3 ± 0.3 days after administration of a first dose consisting of two intramuscular doses of 1.5 mg (approximately 3.75 μg per kg of body weight) of cetrorelix. In the examples shown herein, in alpacas, a new FWE is observed at a fixed time approximately 7.92 ± 0.24 days after administration of a first dose consisting of two intramuscular doses of 1.5 mg of cetrorelix acetate, or approximately 5.33 ± 0.70 days after a single intramuscular injection of 1.5 mg of cetrorelix.

[0100] In the methods described herein that involve a superovulation or hyperstimulation method, the step of "administering an effective amount of FSH or an FSH agonist to a female mammal at regular intervals after administration of a GnRH antagonist" means that the administration of FSH or an FSH analog is initiated around the time point of the predicted FWE (resulting from the administration of the GnRH antagonist) and then continued for 3 to 7 days depending on the mammalian species. In one embodiment, FSH can be administered by intramuscular injection at 12-hour intervals for 4 or 7 days starting from the time point of FWE. As will be appreciated, FSH can block the regression of existing dominant follicles. Thus, for the purpose of inducing the emergence of a new wave and / or synchronizing the emergence of follicular waves to induce superovulation or hyperstimulation, FSH should not be co-administered with a GnRH antagonist. In the methods described herein, FSH should be administered only after the levels of endogenous LH and / or FSH have been reduced to basal levels and / or after all existing dominant follicles are expected to have regressed after administration of the GnRH antagonist and a new follicular wave has been induced.

[0101] As is understood in the art, in the case of Ichiro, for example, in species that are spontaneous ovulating animals (e.g., bovines, sheep, horses, pigs, dogs), individual mammals within a population can have existing corpora lutea that can interfere with synchronization. Thus, additional treatments can be performed to avoid the need to examine individual animals for the presence of existing functional corpora lutea. For example, a) to prevent the LH surge and ovulation, b) to prime the endometrium to prevent early post-ovulatory PGF release, and c) since progesterone withdrawal triggers the ovulatory cascade, progesterone can be co-administered with a GnRH antagonist. Similarly, PGF can be administered around the time of progesterone withdrawal to induce regression of all existing corpora lutea, which further decreases plasma progesterone levels and, if a dominant follicle in the growing or early quiescent phase is present at that time, triggers the ovulatory cascade. Thus, in the methods described herein, the step of "administering prostaglandin (PGF) or a prostaglandin analogue at a timed interval after administration of a GnRH antagonist" or the step of "administering prostaglandin (PGF) or a prostaglandin analogue at a timed interval after administration of FSH" means administering PGF or a PGF analogue around the time when functional corpora lutea become sensitive to luteolysis induced by PGF. Similarly, the administration of progesterone or a progesterone analogue is discontinued around the time when induction of the ovulatory cascade is desired (e.g., when a dominant follicle in the growing or early quiescent phase is expected to be present after FWE resulting from administration of a GnRH antagonist). For example, in the case of heifers administered a single intramuscular dose of 20 μg of cetrorelix acetate per kg of body weight, PGF or a PGF analogue is administered at a timed interval about 7 - 12 days after administration of the GnRH antagonist, or in some cases about 7 - 8 days after (or about 3 - 7 days after FWE, or in some cases about 3 - 4 days after). Similarly, in beef cattle, progesterone or a progesterone analogue is discontinued about 7 - 12 days after administration of the GnRH antagonist, or in some cases about 7 - 8 days after (or about 3 - 7 days after FWE, or in some cases about 3 - 4 days after).In some cases, progesterone or a progesterone analogue is discontinued about 0 to 48 hours after administration of PGF or a PGF analogue. The appropriate time point for administering PGF or a PGF analogue and / or for discontinuing progesterone or a progesterone analogue can be determined by one of ordinary skill in the art.

[0102] As is understood in the art, ovulation-inducing agents are preferably administered when a dominant preovulatory follicle is present. Thus, in the methods described herein, the step of "administering an effective amount of an ovulation-inducing agent at a regular time after administration of a GnRH antagonist" or the step of "administering an effective amount of an ovulation-inducing agent at a regular time after administration of FSH" means administering the ovulation-inducing agent around the time when a dominant preovulatory follicle has developed after the FWE resulting from the administration of the GnRH antagonist. For example, in beef cattle, the dominant preovulatory follicle is expected to develop about 9 to 14 days after administration of the GnRH antagonist (or about 5 to 7 days after the expected FWE), or about 5 to 9 days after administration of FSH. Thus, the ovulation-inducing agent can be administered about 9 to 14 days after administration of the GnRH antagonist (or about 5 to 7 days after the expected FWE), or about 5 to 9 days after administration of FSH. In the methods described herein, the step of "administering an effective amount of an ovulation-inducing agent at a regular time after administration of PGF or a PGF analog", the step of "administering an effective amount of an ovulation-inducing agent at a regular time after discontinuation of progesterone or a progesterone analog", or the step of "administering an effective amount of an ovulation-inducing agent at a regular time after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog" means administering the ovulation-inducing agent after regression of all existing corpora lutea and / or after the preovulatory / LH surge after administration of PGF or a PGF analog and / or discontinuation of progesterone or a progesterone analog. For example, in the case of heifers, regression of the corpus luteum and / or the preovulatory / LH surge occurs about 2 days after administration of PGF or a PGF analog and / or discontinuation of progesterone or a progesterone analog. Thus, the ovulation-inducing agent can be administered about 2 days after discontinuation of progesterone or a progesterone analog and / or administration of PGF or a PGF analog. The appropriate time point for administering the ovulation-inducing agent can be determined by those skilled in the art.

[0103] In the methods described herein that involve breeding / mating or artificial insemination, "at a fixed time after administration of the ovulation inducer" means that mating or artificial insemination is performed within a certain period from the expected ovulation resulting from the administration of the ovulation inducer, for example, before the expected ovulation, around the time point of the expected ovulation, and / or within a certain period after the expected ovulation. The timing of ovulation can depend on the ovulation inducer, the dosage, the animal species, and the timing of mating or artificial insemination according to the species (before, around, and / or after the time point of ovulation). For example, in alpacas, ovulation occurs approximately 24 to 30 hours after administration of LH (regardless of mating), and alpaca mating or artificial insemination is performed approximately 12 to 24 hours before ovulation. Thus, alpaca mating or artificial insemination can be performed 0 to 18 hours after administration of LH, for example, approximately 12 hours after administration of LH. Mating or artificial insemination can be performed, for example, approximately 12 to 24 hours before ovulation in beef cattle, sheep, goats, and alpacas, or approximately 24 to 36 hours after ovulation in dogs, for example. In beef cattle, mating or artificial insemination is performed up to approximately 1 day after administration of the ovulation inducer, for example, approximately simultaneously with, 12 hours after, and / or 24 hours after administration of the ovulation inducer (e.g., GnRH or LH). As will be understood, the appropriate time point for mating or artificial insemination depends on the mammalian species and can be determined by those skilled in the art.

[0104] In the methods described herein involving oocyte recovery, the step of "recovering one or more oocytes at regular intervals after administration of a GnRH antagonist", or "recovering one or more oocytes at regular intervals after administration of FSH or an FSH agonist", or "recovering one or more oocytes at regular intervals after administration of an ovulation inducer" means that the oocytes are recovered at a time when they are expected to be at the desired stage of development. For example, immature oocytes can be recovered about 2 to 4 days after the expected FWE if FSH is not used, or about 3 to 9 days after FWE if FSH has been administered. Oocytes can be recovered using any suitable method, for example, oocytes (including cumulus-oocyte complexes) and follicular fluid can be aspirated from the ovary using a needle under ultrasonic guidance with the use of a vacuum. For example, in beef cattle, oocytes can be recovered 6 to 11 days after GnRH antagonist treatment without FSH treatment, or 3 to 9 days after the start of FSH treatment, or 30 to 48 hours after progesterone withdrawal or PGF administration, or 0 to 24 hours after intramuscular injection of an ovulation inducer such as GnRH or LH. As will be understood, the appropriate time point for oocyte recovery depends on the mammalian species and the desired stage of development of the oocytes and can be determined by those skilled in the art.

[0105] In the methods described herein involving embryo recovery, the step of "recovering one or more embryos at regular intervals after artificial insemination", or "recovering one or more embryos at regular intervals after mating" means that the embryos are recovered when they are expected to be at the morula or blastocyst stage. Embryos can be recovered from the uterus using any suitable method, for example, using a surgical method or non-surgically by placing a catheter into the uterus or uterine horn through the vagina and cervix. Embryos can be recovered, for example, about 5 to 10 days after artificial insemination. For example, in beef cattle, embryos are recovered 7, 8, and / or 9 days after artificial insemination. As will be understood, the appropriate time point for embryo recovery depends on the mammalian species and the desired stage of development of the embryos and can be determined by those skilled in the art.

[0106] In the methods described herein that involve embryo transfer, the step of "transplanting the embryo into the female mammal at a fixed time after administration of the ovulation inducer" means that the embryo is transplanted after ovulation and subsequent development of the corpus luteum that produces progesterone, which can be, depending on the species, for example, about 2 to 8 days after the expected ovulation time, or about 3 to 10 days after administration of the ovulation inducer. Any suitable embryo can be transplanted, including fresh / unfrozen or cryopreserved embryos recovered in vivo or produced in vitro. The embryo can be transplanted using any suitable method known in the art, for example, the embryo can be transplanted into the uterus or uterine horn surgically or non-surgically by placing a catheter through the vagina and cervix into the uterus or uterine horn. The embryo can be transplanted into the recipient, for example, about 3 to 10 days after administration of the ovulation inducer, depending on the species and the stage of the embryo. For example, in beef cattle, the embryo can be transplanted 7, 8, or 9 days after administration of LH, GnRH, or estradiol. As understood in the art, progesterone (e.g., produced by the corpus luteum) acts on the endometrium, which develops in synchrony with all developing embryos, such that the embryo age matches the number of days after ovulation (e.g., within about 24 hours). For example, if the embryo is transplanted into the recipient about 7 days after ovulation, the embryo will be at about 7 days of development. For example, in humans, the fertilized egg can be transplanted directly into the uterus 2 days after ovulation, and the blastocyst can be transplanted into the uterus 5 to 6 days after ovulation. Thus, it should be understood that the appropriate timing of embryo transfer depends on many factors, including the mammalian species, the ovulation inducer, and the stage of the embryo to be transplanted, and can be determined by those skilled in the art.

[0107] As used herein, the term "mammal" is properly used to refer to animal species in which female animals have an ovulation cycle (e.g., estrus). Unless otherwise clearly indicated from the context, the term "animal" is also used herein to refer to mammals, and in some contexts, is used specifically to refer to female mammals, including humans. The term "domestic animal" refers to mammalian species that are generally bred or maintained in captivity, such as livestock / carrier animals (e.g., Bovinae animals such as cattle and buffalo, Camelidae animals such as llamas, alpacas, and camels, sheep (e.g., sheep), goats (e.g., goats), pigs (e.g., pigs), horses (e.g., horses and donkeys), etc.), as well as pets (e.g., cats, dogs), or specifically, female animals of these species. Similarly, the term "wild animal" includes semi-domesticated mammalian species and non-domesticated mammalian species, such as bison, elk, caribou, deer (e.g., white-tailed deer), bighorn sheep, non-human primates, canines, and felines, such as tigers, lions, leopards, or female animals of these species, etc. In one embodiment, the mammal is a domestic animal, optionally a Bovinae animal, a Camelidae animal, a sheep, a goat, a pig, a canine, or a feline. In one embodiment, the mammal is a wild animal, optionally a bison, an elk, a caribou, a deer, a bighorn sheep, a non-human primate, a canine, or a feline. In one embodiment, the mammal is a human.

[0108] As used herein, the term "mixed population" means a population of female mammals whose stages of ovulation cycle are synchronized.

[0109] As used herein, the term "oocyte" means a female germ cell at any stage that can give rise to an embryo upon fertilization, including, but not limited to, oocytes, eggs, and cumulus-oocyte complexes (e.g., oocytes surrounded by small or large layers of granulosa / cumulus cells).

[0110] As used herein, the term "embryo" means an animal at an early stage of development, such as a fertilized egg, a morula, or a blastocyst.

[0111] As used herein, the terms "administered" or "administering" mean administering to an animal an effective amount of a compound or composition of the present disclosure. The terms "co-administered" or "combination therapy" mean administering to an animal at least two compounds or compositions such that an effective amount or effective concentration of each of the two or more compounds is present in the animal at a given time point. The compounds according to the present disclosure can be co-administered to an animal simultaneously, but the term encompasses both simultaneous administration of two or more agents and administration at different time points, so long as an effective concentration of all co-administered compounds or compositions is present in the animal at a given time point.

[0112] As used herein, the expression "effective amount" means an amount effective at the time of dosing and over the period necessary to achieve the desired result. For example, in the context of follicular wave emergence (FWE), an effective amount is an amount that induces FWE in female mammals at approximately the same time (or within a consistent time frame, e.g., within (±) 1 or 2 days) after administration as compared to the response obtained without administering the compound. The effective amount can vary according to 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 the given drug or compound, pharmaceutical formulation, route of administration, mammalian species being treated, etc.

[0113] The GnRH antagonist can be administered according to an appropriate schedule, e.g., depending on the given drug or compound, formulation, route of administration, and / or mammalian species, as a single dose, or as multiple doses (e.g., 2 doses) separated by an appropriate period (e.g., about 1 day), or as any other appropriate number of doses and / or period. In one embodiment, the GnRH antagonist is administered as a single dose. In one embodiment, the GnRH antagonist is administered as 2 doses at about 1-day intervals. In one embodiment, the GnRH antagonist is administered as a single dose in a dosage form that provides sustained or continuous release over a period of time, e.g., about 1 day, about 2 days, about 3 days, or more days.

[0114] GnRH antagonists can be administered by any suitable route of administration and using any suitable dosage form, for example, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and media, for parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, inhalation or spray (e.g., via aerosol), mucosal administration, rectal administration, vaginal administration, skin patch or topical application to the skin, or oral administration.

[0115] Compositions containing GnRH antagonists such as cetrorelix can be provided in any suitable dosage form. The term "dosage form" as used herein refers to the physical form of administration, for example, containing the compounds of the present disclosure, and includes, but is not limited to, injectable dosage forms such as sterile solutions and sterile powders appropriately formulated for injection, for example, including those for reconstitution; and liquid and solid dosage forms including, for example, tablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets, lozenges, elixirs, suspensions, syrups, wafers, re-suspendable 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 that releases the compound in a controlled and consistent manner over a period of time, for example, over several days or weeks. Methods for making depot injections are described, for example, in U.S. Patent No. 3,089,815, the entire disclosure of which is incorporated herein by reference under the title "Injectable pharmaceutical preparation, and a method of making same". Dosage forms can include, for example, dosage forms suitable for mucosal administration, including gels, creams, ointments, foams, tablets, or capsules, or dosage forms via inserts including substrates (e.g., silicone coatings, tampons, or sponges) coated with or impregnated with GnRH antagonists, for example, suitably for vaginal administration or skin patches.

[0116] Examples of suitable dosage ranges for cetrorelix include, when administered by intramuscular injection, for example, from about 2 μg per kg of body weight to about 200 μg per kg of body weight, from about 5 μg per kg of body weight to about 100 μg per kg of body weight, from about 7.5 μg per kg of body weight to about 75 μg per kg of body weight, from about 20 μg per kg of body weight to about 50 μg per kg of body weight, or any suitable dosage or dosage range between from about 2 μg per kg of body weight to about 200 μg per kg of body weight, for example, about 2.5 μg per kg of body weight, about 3 μg per kg of body weight, about 4 μg per kg of body weight, about 5 μg per kg of body weight, about 7.5 μg per kg of body weight, about 10 μg per kg of body weight, about 15 μg per kg of body weight, about 20 μg per kg of body weight, about 25 μg per kg of body weight, about 30 μg per kg of body weight, about 35 μg per kg of body weight, or about 40 μg per kg of body weight may be included. In one embodiment, the effective dose of cetrorelix is about 7.5 μg per kg of body weight, administered by intramuscular injection. In one embodiment, the effective dose is about 40 μg per kg of body weight, administered by intramuscular injection. The dosage can be administered as a single dose or as multiple doses spaced apart by a certain period, for example 24 hours or the like. In one embodiment, about 7.5 μg of cetrorelix per kg of body weight is administered as a single intramuscular dose. In one embodiment, about 7.5 μg of cetrorelix per kg of body weight is administered as two intramuscular doses of about 3.75 μg per kg of body weight at about 24-hour intervals. In one embodiment, about 20 μg of cetrorelix per kg of body weight is administered as a single intramuscular dose. In one embodiment, about 40 μg of cetrorelix per kg of body weight is administered as two intramuscular doses of about 20 μg per kg of body weight at about 24-hour intervals.

[0117] GnRH antagonists can be administered or used in combination with one or more additional drugs, for example, at regular intervals after administration of the GnRH antagonist, to provide enhanced or additional reproductive management control for inducing ovulation (which can be performed synchronously in a mammalian population, thereby synchronizing ovulation). Controlled or synchronized ovulation is applied in breeding management, timed artificial insemination protocols, or embryo transfer protocols. For example, GnRH antagonists can optionally be used in combination with progesterone or a progesterone analog, followed by administration of prostaglandin (PGF) or a PGF analog and / or followed by administration of an ovulation inducer to delay and / or enhance synchronization of ovulation following the follicular wave induced by the GnRH antagonist. Such combinations may be particularly suitable for timed artificial insemination or embryo transfer protocols. Thus, in one embodiment, the method further comprises the step of administering an effective amount of one or more additional drugs. In one embodiment, the one or more additional drugs include progesterone or a progesterone analog, prostaglandin (PGF) or a PGF analog, and / or an ovulation inducer. 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, for timed AI and embryo transfer, the use of FSH, the dose of PFG2a, and LH / GnRH are different. In oocyte retrieval, LH can be omitted.

[0118] Progesterone and progesterone analogs can be administered using progesterone drug delivery devices, including products such as controlled internal drug release (CIDR) devices (Zoetis), PRID™ (CEVA), cue-mate™ (Vetoquinol), and subcutaneous devices such as syncromate-B. Oral supplementation with progesterone analogs such as norgestomet, i.e., melengestrol acetate (MGA), can also be used in combination with GnRH antagonists.

[0119] Prostaglandins (PGF) and PGF analogs include, but are not limited to, prostaglandin 2 alpha (PGF2a), cloprostenol, dinoprost, dinoprost tromethamine, bimatoprost, travoprost, carboprost, and latanoprost.

[0120] As used herein, "ovulation inducer" means an agent that, when administered to a female mammal, causes ovulation of any pre-ovulatory existing dominant follicles present in the female mammal. Ovulation inducers include, but are not limited to, GnRH and GnRH analogs and agonists (e.g., buserelin, deslorelin, fertirelin, gonadorelin, goserelin, leuprolin, triptorelin), LH and LH analogs, agonists, conjugates, and recombinant products (e.g., human chorionic gonadotropin / hCG, equine chorionic gonadotropin / eCG), and estradiol and estradiol esters, analogs, and agonists (e.g., estradiol-17 beta, estradiol benzoate, estradiol valerate, estradiol cypionate). Appropriate ovulation inducers can be selected by one of ordinary skill in the art and can vary depending on the mammalian species. For example, appropriate ovulation inducers for camels include GnRH or LH, but not estradiol.

[0121] III. Devices and Kits One aspect includes a drug delivery device containing a GnRH antagonist. Suitably, the drug delivery device is configured to provide a controlled release of an effective amount of the GnRH antagonist over a period of time, such as from about 1 day to about 3 days, etc. In one embodiment, the GnRH antagonist is cetrorelix. In one embodiment, the drug delivery device further contains progesterone and is configured to provide a controlled release of an effective amount of progesterone over a longer period of time, such as at least about 5 days to at least about 10 days or more, and in some cases, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more days.

[0122] One aspect includes a kit containing a GnRH antagonist, a suitable container or packaging, and / or instructions for use of the GnRH antagonist for use, for example, in a timed protocol, such as a timed artificial insemination protocol, a timed oocyte retrieval protocol, or a timed embryo production or embryo transfer protocol, for synchronization of FWE and optionally ovulation in a population of female mammals, or for use in a gonadotropin stimulation and superovulation treatment protocol for embryo production / transfer in some cases. In one embodiment, the GnRH antagonist is provided within a drug delivery device. In one embodiment, the kit further includes an applicator or other suitable delivery device. In one embodiment, the kit further includes one or more additional drugs, such as progesterone, PGF2A, or analogs thereof, such as cloprostenol, GnRH, eCG, and / or LH, etc.

Examples

[0123] III. Examples The following non-limiting examples are illustrative of the present application.

[0124] (Example 1) Effect of GnRH antagonist (cetrorelix) on response to kisspeptin treatment Kisspeptin is a neuropeptide product of the kiss-1 gene that is cleaved and / or degraded into peptides consisting of 54 amino acids, 14 amino acids, 13 amino acids, and 10 amino acids (Kotani et al., 2001). GnRH and kisspeptin immunoreactive cells are located in close association with the preoptic area and arcuate nucleus of the hypothalamus in mice, primates, sheep (Ramaswamy et al., 2011; Smith et al., 2008; Clarkson and Herbison, 2006), and beef cattle (Tanco et al., 2016). Peripheral injection of kisspeptin induces the secretion of luteinizing hormone (LH) and ovulation in several mammalian species, including beef cows (Leonardi et al., 2019; Leonardi et al., 2018) and seasonally anovulatory ewes (Caraty et al., 2007) (Caraty et al., 2007; Caraty et al., 2013; d'Anglemont de Tassigny et al., 2008; Ezzat Ahmed et al., 2009; Matsui et al., 2004). Furthermore, intravenous administration of kisspeptin-10 induces the release of GnRH into the pituitary portal circulation in sheep (Smith et al., 2011). In beef cattle, it is not clear whether the 10-amino acid kisspeptin fragment (kisspeptin-10) can cross the blood-brain barrier and stimulate GnRH neurons after peripheral injection.

[0125] It is not yet clear whether peripheral kisspeptin can activate GnRH neurons in beef cattle.

[0126] Endogenous and exogenous GnRH induce the release and synthesis of LH from the pituitary gland in beef cattle (Vizcarra et al., 1997; Yoshioka et al., 2001), and administration of a GnRH antagonist prevents the LH surge (Ginther et al., 2012). Peripheral administration of kisspeptin 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 previous studies, a progressive increase in LH secretion was detected within 15 minutes after repeated intravenous administration of kisspeptin-10 in cows with plasma progesterone below 1.7 ng / mL (Leonardi et al., 2019). However, the mechanism by which peripheral administration of kisspeptin-10 induces LH release in cows is not clear. In vitro studies in horses and beef cattle have suggested the possibility of direct LH release from anterior pituitary cells by kisspeptin (Ezzat et al., 2010; Magee et al., 2020). In vivo, whether kisspeptin-10 crosses the blood-brain barrier to stimulate GnRH neuron cell bodies or acts on GnRH nerve terminals in the median eminence, and whether the observed LH release is due to a direct effect on pituitary gonadotropin-secreting cells, remains unclear.

[0127] Objective: To determine whether pretreatment with a GnRH antagonist alters the pattern of LH release and ovulation induced by kisspeptin. Cetrorelix was used as an inhibitor of kisspeptin action in a dominant follicle model that could be maintained under low plasma progesterone.

[0128] Hypothesis: 1) In beef cattle, pretreatment with a GnRH antagonist (cetrorelix) before kisspeptin treatment suppresses LH release and prevents ovulation.

[0129] Method: The human kisspeptin-10 (hKP-10) peptide (YNWNSFGLRF-NH2, SEQ ID NO: 1) was custom synthesized by GenScript USA Inc., Piscataway, NJ, USA, with >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 number AY117143) and has been used previously in beef cattle. The peptide was pre-tested for solubility and dissolved in ultrapure water at 10 mg / mL.

[0130] The study was conducted in Hereford-crossed heifers (n = 15, body weight 500 ± 24 Kg, 17 - 18 months of age), which had a corpus luteum in one of their ovaries at the start of the experiment. The heifers were maintained in outdoor pens at the Goodale Research Farm, University of Saskatchewan (52°N, 106°W). The heifers were fed barley silage and had free access to hay and water. Throughout the study period, the heifers had access to mineral blocks. All procedures were conducted in accordance with the Canadian Council on Animal Care and were approved by the Protocol Review Committee of the University of Saskatchewan.

[0131] A schematic diagram of the experimental design is shown in Figure 1. Heifers (n = 15) were selected from a larger group based on the detection of the corpus luteum (CL) by transrectal ultrasonography. Immediately after ultrasonography, PGF2α was administered to the heifers twice at 12-hour intervals. The previously described low progesterone bovine subfamily animal model (Leonardi et al., 2019) was used to test the hypothesis. Briefly, the ovaries were examined once a day by ultrasonography to detect ovulation. Three days after ovulation, follicles with a diameter of ≥5 mm in both ovaries were ablated by aspiration of the follicles under transvaginal ultrasound guidance, and the appearance of a new follicular wave was induced 1.5 days after this procedure (Bergfelt et al., 1994). After follicular ablation, a progesterone device was placed intravaginally (CIDR, Zoetis). The day when the wave appeared (day 0) was defined as the day when the follicles of the wave (diameter 4 to 5 mm) with an increase in diameter were first recorded. PGF2α was administered im to the heifers on days 3.5 and 4 (i.e., 8 days and 8.5 days after ovulation) to regress the corpus luteum, but the CIDR device was left in place until day 12 to maintain a low progesterone environment sufficient to prevent natural ovulation. On day 6, an indwelling jugular vein catheter was placed as described (Bergfelt et al., 1997), and the heifers were randomly assigned to three groups (n = 5 per group): 1) three iv administrations of 15 mg of human kisspeptin-10 at 1-hour intervals (Kp10 group), 2) pretreatment with a single im administration of cetrorelix acetate (20 μg per kg body weight, diluted in 5% D-mannitol w / v, GnRH antagonist - Sigma #C5249, Sigma #M4125), and three iv administrations of 15 mg of human kisspeptin-10 at 1-hour intervals 3 hours later (cetrorelix group), or 3) three iv administrations of normal saline at 1-hour intervals (control group). After treatment (day 6), ovarian ultrasonography was performed once a day until the end of the experiment to detect ovulation of the existing dominant follicle (i.e., the dominant follicle that was present at the time of treatment) or the dominant follicle arising from the next wave. The progesterone device was discontinued on day 12 (i.e., 13 days after insertion), and ultrasonography was continued until ovulation was detected.

[0132] Blood samples and hormone assays: Serial blood samples were collected at 15-min intervals from -30 to 150 min (0 min = time of first hKP-10 or saline injection) on the treatment day using an indwelling jugular catheter. All blood samples were collected in heparinized tubes (Vacutainer, BD, Franklin Lakes, NJ, USA). Immediately after sampling, the tubes were centrifuged at 1500 × g for 15 min to separate plasma and store at -20 °C.

[0133] Plasma samples from both experiments were analyzed for LH and progesterone concentrations in the laboratory of Dr. O. J. Ginther at the University of Wisconsin (Madison, WI, USA). Plasma LH concentrations in cattle were measured by a validated radioimmunoassay (Bolt et al., 1990) with modifications as reported (Ginther et al., 1999). Briefly, 125 LH concentrations were measured in duplicate using USDA-bLH-B-6 for 1-iodination and preparation of the reference standard and 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 interassay coefficients of variation and the average sensitivity were 6.23%, 12.24%, and 0.03 ng / mL, respectively. Progesterone concentrations were measured using antibody-coated tubes and 125 A commercial solid-phase RIA kit with I-labeled progesterone (ImmuChem Coated Tube Progesterone 125 RIA kit, MP Biomedical, Costa Mesa, CA) was used in a single assay batch as described (Ginther et al., 2005). The intra-assay coefficient of variation and sensitivity to progesterone were 11.97% and 0.06 ng / mL, respectively.

[0134] Statistical analysis: Data analysis was performed using SAS (Statistical Analysis System, software package 9.4, SAS Institute Inc., Cary, NC, USA). In both experiments, one-way analysis of variance was used to analyze single-point measurements (i.e., the diameter of the dominant follicle at the time of treatment and 24 hours after treatment, the diameter of the CL at the time of treatment, and the progesterone concentration at the time of treatment). A P-value of ≤0.05 was assumed to be statistically significant, while a P-value between >0.05 and ≤0.1 was assumed to show a tendency of difference. When the P-value of the test detected a difference, Tukey's post hoc test was used for multiple comparisons. The ovulation rate was analyzed using the GLIMMIX procedure.

[0135] Analysis of repeated measurement data (e.g., plasma LH levels, follicle dynamics) was performed using the MIXED model procedure, in which treatment, time, and the interaction between treatment and time were tested, and the repeat statement was included in the syntax (repeated days subject=cowID). The initial analysis tested five covariance structures (SIMPLE, CS, AR(1), ANTE(1), or UN), and the model with the minimum AICC value was selected for the final analysis. All values are reported as mean ± SEM.

[0136] Results: The diameter of the dominant follicle at the time of treatment (day 6 of the follicular wave) and 24 hours after treatment did not differ among the Kp10 group, cetrorelix group, and control group (Table 1). Plasma progesterone concentrations at the time of treatment were similar among the groups. The Kp10 group had higher plasma LH concentrations than the cetrorelix group and the control group (P<0.001, Figure 2B). Plasma LH concentrations remained higher in the Kp10 group than in the other two groups from 15 to 240 minutes (0 minutes = first injection at treatment, Figure 2A). Ovulation of the existing dominant follicle was recorded in 4 out of 5 heifers in the Kp10 group, compared with 0 out of 5 heifers in the cetrorelix group and the control group (Table 1). A new follicular wave immediately appeared in all 4 heifers that ovulated after kisspeptin treatment (Figure 2A). Regression of the existing dominant follicle was recorded in the cetrorelix group 3 to 4 days after treatment (Figure 2A). The appearance of a new follicular wave occurred 3.40±0.75 days after treatment in the cetrorelix group (i.e., without ovulation of the existing dominant follicle), compared with 2.75±0.75 days after treatment in the Kp10 group (in 4 out of 5 heifers, after ovulation of the existing dominant follicle). In the control group, the existing dominant follicle did not regress and ovulated on day 15 after CIDR removal (which was discontinued on day 12). All new dominant follicles in the Kp10 group (n = 4) and the cetrorelix group (n = 5) ovulated on day 15 after CIDR removal (day 12).

[0137]

Table 1

[0138] In all 5 out of 5 heifers in the cetrorelix+KP10 group, the appearance of a new wave occurred 3.40±0.75 days after treatment, during the mid-dormancy of the dominant follicle, under a low progesterone environment.

[0139] The appearance of the wave was due to the regression of the existing dominant follicle caused by low plasma LH levels. The dominant follicles of the new wave were able to ovulate after CIDR removal.

[0140] In all of the heifers in the control group treated with physiological saline (n = 5), the dominance of the existing follicles was maintained and no new wave appeared. When the CIDR device was removed on day 12, the existing dominant follicles were able to ovulate.

[0141] In 4 out of 5 heifers in the KP10 group, the appearance of a new wave occurred 2.75 ± 0.75 days after treatment, due to the ovulation of the existing dominant follicle (resulting from an increase in plasma LH levels). The CL resulting from the ovulation of the existing dominant follicle had a short lifespan.

[0142] Conclusion: Unexpected downstream effects of LH suppression in cetrorelix-treated heifers were also seen on the dominant follicles, and in this group, none of the heifers were able to ovulate (0 out of 5 ovulated), whereas 4 out of 5 heifers ovulated by 48 hours after kisspeptin treatment. Interestingly, the dominant follicles in all animals entered the regressive phase by 72 hours after treatment with kisspeptin and GnRH antagonist.

[0143] In conclusion, pretreatment with the GnRH antagonist cetrorelix, 3 hours before the start of intravenous treatment with kisspeptin, suppressed the LH surge induced by kisspeptin, prevented ovulation, and regressed the existing dominant follicles in all 5 heifers, and then a new follicular wave appeared on average 3.4 days after treatment. A similar mechanism of LH release via kisspeptin is downstream of GnRH synthesis and is probably due to the induction of the release of prepro-GnRH from the nerve terminals in the median eminence.

[0144] (Example 2) Study of alpaca cetrorelix at known stages Setrorelix inhibits the preovulatory LH surge induced by the ovulation-inhibiting factor (OIF) in llamas, suggesting that LH secretion is regulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus (Silva et al., 2011). It is not clear whether the effects seen in the dominant follicle in Example 1 apply to llamas. Furthermore, it is not clear whether the same effects are seen at other stages of follicular development (preovulatory growth, quiescence, regression) as well as luteal development (metestrus, diestrus, and proestrus).

[0145] Objective: To identify the pattern of growth of the dominant follicle in camelids after treatment with a GnRH antagonist at various stages of the follicular wave.

[0146] Hypothesis: 1) GnRH antagonist causes regression of both the dominant follicle in the early / mid-growth phase and mid-quiescence phase, and 2) a new wave appears at the same time point after treatment regardless of the follicular status of the dominant follicle.

[0147] Methods: The specific objective of this experiment was to identify the pattern of growth of the dominant follicle in alpacas after treatment with a GnRH antagonist at two contrasting stages of the follicular wave. Based on data on natural waves in alpacas, the dominant follicle was expected to be in the early to mid-growth phase (6 to 7 mm in diameter) on day 5 and in mid-quiescence (9 to 10 mm) on day 10, and therefore treatment was planned to start on these days.

[0148] The experimental protocol is illustrated in Figure 3. Transrectal ovarian ultrasonography was performed using an Esoate My Lab 5 ultrasound device and a 7.5 linear array transducer attached to a custom-made rigid PVC pipe handle. The pipe was slightly curved at the end of the transducer to facilitate transducer manipulation. The transducer and plastic handle were lubricated with methylcellulose ultrasound gel and gently inserted into the rectum after defecation. The reproductive tract and ovaries were positioned, and 10- to 20-second video clips of the left and right ovaries were recorded. The ovaries were sketched at the time of imaging once a day to record the position, size, and number of follicles >2 mm in diameter, and the size and position of the corpus luteum (if present). Ultrasonography was performed once a day. Recording of experimental data was initiated when a new follicular wave (defined as an existing follicular wave) was detected in the animals naturally. The day on which the wave appeared was designated as day 0, and retrospectively defined as the first detection of a dominant follicle of 4 or 5 mm size, accompanied by an increase in the number of 3- to 4-mm follicles.

[0149] Based on the day on which the wave appeared, alpacas were assigned to one of three treatment groups (n = 7 animals per group) using a systematic random sampling method. The first alpaca group (control group) was not treated, and their ovaries were examined once a day. Blood samples were obtained once a day from day 4 to day 12, and hormonal data were compared on approximately the same day after cetrorelix treatment. The second alpaca group (D5 group) received a first intramuscular injection of 1.5 mg (0.5 mg per mL of solution in 3 mL) of cetrorelix acetate on day 5 and a second injection of 3 mL on day 6 (i.e., two i / m injections of 1.5 mg of cetrorelix at 24-hour intervals for a total dose of 3 mg). The dose of cetrorelix administered was approximately equal to 20 μg per kg of body weight per day (considering the average body weight of alpacas = 75 kg) and was based on previous studies in llamas and beef cattle (Silva et al., 2011; Ulker et al., 2001). Alpacas in the third group (D10 group) received a first intramuscular injection of 1.5 mg of cetrorelix acetate on day 10 and a second injection of 1.5 mg on day 11.

[0150] The cetrorelix treatment was performed during the existing follicular wave. The changes in the size of the dominant follicle, the largest subordinate follicle, and the number of follicles ≥ 3 mm were recorded once a day from the natural appearance of the existing follicular wave until the 6th day after the appearance of the next (post-treatment) follicular wave. The inter-wave interval was determined based on the period between the appearances of consecutive follicular waves.

[0151] Blood samples (5 to 8 mL) were collected via jugular vein puncture into heparinized test tubes (BD Vacutainer, BD, Franklin Lakes, NJ, USA). The blood samples were maintained on an ice pack until centrifugation at 200 rpm (within 1 hour of collection), and plasma samples were obtained by centrifugation. The plasma samples were immediately frozen at -20°C until measurement of LH in the plasma. Plasma samples were obtained on days 4 to 7 (i.e., 1 day before to 1 day after the cetrorelix treatment) in group 2 and on days 9 to 12 in group 3. For direct comparison, plasma samples were obtained on days 4 to 12 in the control group.

[0152] Results: The results are shown in Table 2. The post-treatment data of one animal in the D10 group were excluded because ovulation started on the 10th day before treatment.

[0153] In the untreated control group (n = 7), the appearance of the next wave occurred on day 14.57 ± 0.81 after the appearance of the first wave, i.e., the inter-wave interval of the existing wave ranged from 13 to 19 days, and the median was 14 days. On average, the dominant follicle entered the quiescent phase on day 7.6 ± 0.55 (data combined from the control group and D10 group).

[0154] The mean onset day of dominant follicle regression was earlier in the D5 group than in the D10 group and the control group (day 8.7 ± 0.89, day 12.9 ± 0.34, day 13.6 ± 0.90, P < 0.001), and the diameter of the dominant follicle was smaller in the D5 group than in the D10 group and the control group (7.4 ± 0.5 mm on day 6.0 ± 0.90, 10.9 ± 0.70 mm on day 10.3 ± 0.68, 10.2 ± 0.93 mm on day 10.0 ± 0.90, P = 0.005).

[0155] The duration of the resting phase of the dominant follicle was 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).

[0156] The inter-wave interval was short in the D5 group, intermediate in the control group, and long in the D10 group (12.7 ± 0.36 days, 14.57 ± 0.81 days, 17.71 ± 0.52 days, P < 0.001).

[0157] When groups 2 and 3 were combined, wave emergence occurred on average 7.92 ± 0.24 days after the first cetrorelix injection (range 7 to 9 days).

[0158] Taking all data together, when cetrorelix treatment was performed on day 5 of the wave (n = 7), the dominant follicle ceased growth, had a shorter resting phase than the control or D10 groups, reached maximum diameter earlier, and the onset of regression occurred earlier. A new wave was induced 7.71 ± 0.36 days later (day 12.7 of the existing wave, i.e., earlier than the control group).

[0159] When cetrorelix treatment was performed on day 10 of the wave, the dominant follicle regressed simultaneously with the control, and a new wave started 8.17 ± 0.31 days after cetrorelix treatment (day 18.17 of the existing wave, i.e., later than the control group).

[0160] The synchrony of wave emergence (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]).

[0161]

Table 2

[0162] Conclusion: In alpacas, when treatment was initiated during the growing or late quiescent phase of the dominant follicle, the dominant follicle regressed after two cetrorelix injections, and the emergence of the next wave was synchronized.

[0163] (Example 3) Study on random days in alpacas Objective: To examine the effect of a single injection of cetrorelix on the regression of the dominant follicle and the synchronization of the emergence of the next wave in alpacas when treatment is initiated on random days of the wave.

[0164] Hypothesis: A single treatment with a GnRH antagonist was performed to cause regression of the dominant follicle at a random stage of development and to re-examine whether a new wave would appear at the same time after treatment regardless of the stage of dominance (i.e., similar to Example 2).

[0165] Method: The method of ultrasonography and the recording of follicle endpoints were the same as those described in Example 2. Follicle endpoints were obtained from alpacas (n = 15) before and after treatment, and thus each animal served as its own control. A fixed calendar date (July 16) was pre-assigned to the cetrorelix treatment. From May 31 to June 16, transrectal ultrasonography was performed once a day (n = 15 alpacas). This examination period was used to record the variance in the emergence of waves from the fixed calendar date (May 31) when no treatment was performed (control period). The animals were at the start of this 17-day time window (i.e., longer than the interval between one natural wave) at an unknown / random day of the wave. Thus, all animals were expected to have the emergence of at least one wave that included this period. All alpacas were treated with a single intramuscular injection of 1.5 mg (3 mL) of cetrorelix acetate on July 16, and transrectal ultrasonography was continued once a day from the day of treatment until 3 days after the emergence of the next (post-treatment) wave (treatment period). The synchronization of wave emergence was compared between the control period and the treatment period.

[0166] Statistical analysis: Proc Mixed repeated measures analysis of variance (SAS version 9.4) was performed to compare a series of data (changes in follicle size and number of follicles over several days), and single-point numerical measurements were taken using ANOVA or paired t-tests. Count data were compared using Fisher's exact probability test. The synchrony of wave appearance was compared using Bartlett's and Levene's tests for equality of variances.

[0167] Results: The results of Example 3 are shown in Table 3 to Table 6.

[0168]

Table 3

[0169] The appearance of the next wave occurred on average 5.33 ± 0.70 days after a single treatment with 1.5 mg of cetrorelix on random days of the wave, compared to 8.93 ± 1.21 days without treatment (during the control period).

[0170]

Table 4

[0171]

Table 5

[0172]

Table 6

[0173] The variation (variance 7.38) during the period after cetrorelix treatment was smaller than that during the control period (22.067), indicating synchrony of wave appearance [Bartlett's test for equality of variances, p = 0.049, Levene's test, p = 0.025].

[0174] The range of the confidence interval was 3.8 to 6.8 days after cetrorelix treatment, whereas it was 6.3 to 11.5 days in the control CI.

[0175] (Example 4) Effect of cetrorelix on pre-selection, growing, and regressing dominant follicles in beef heifers Specific objectives: The first objective was to determine the growth patterns of the dominant and first subordinate follicles in beef cattle after treatment with the GnRH antagonist cetrorelix during the pre-selection period (days 1 - 2), during the growth phase of the follicular wave (days 3 - 4), and during the late quiescence / early regression stage (days 6 - 7). The second objective was to record the changes in CL function and progesterone production.

[0176] Hypothesis: Treatment with the GnRH antagonist results in loss of follicular dominance and emergence of a new follicular wave at a consistent and predictable interval after treatment, regardless of the follicular stage at the time of treatment (i.e., pre-selection, growing dominant follicle, late quiescence / early regression).

[0177] Experimental design: A schematic diagram of the experimental design is shown in Fig. 4. The study was conducted at the Livestock and Forage Centre of Excellence (LFCE) at the University of Saskatchewan in pubertal Hereford crossbred beef heifers (n = 32). Puberty was confirmed by the presence or absence of a corpus luteum (CL) via the first transrectal ultrasonography examination. Animals were also weighed at this time point. Animals with a functional CL were given an intramuscular (im) injection of prostaglandin F2α analogue (cloprostenol) to induce CL regression and ovulation. Ultrasonography examinations were performed once daily to detect ovulation and to record the number, size, and location of follicles and CLs in both ovaries. The day on which the follicular wave appeared was initially estimated by a sharp increase in the number of follicles of 3–4 mm and retrospectively confirmed as the day on which the dominant follicle of 4–5 mm size was first detected (defined as day 0). Follicular and hormone data (see the next section for endpoints) were collected from all animals over one complete ovulatory interval.

[0178]

Table 7

[0179] Animals were randomly divided into four groups based on the day of ovulation. The heifers in group 1 (control group) were administered 3 mL of normal saline (sham treatment negative control; n = 3 on days 1 and 2, n = 3 on days 3 and 4, n = 2 on days 6 and 7). The heifers in group 2 were administered 1.5 mg of cetrorelix on days 1 and 2 (pre-selection phase of the dominant follicle, n = 8), the heifers in group 3 were administered cetrorelix on days 3 and 4 (growing phase of the dominant follicle, n = 8), and the heifers in group 4 were administered cetrorelix on days 6 and 7 (late quiescence / early regression phase of the dominant follicle, n = 8). The dose of cetrorelix (1.5 mg per treatment), the route of administration (intramuscular), and the duration of treatment (i.e., two doses at 24-hour intervals) were selected based on Example 2 and previous studies in beef cattle (Ulker et al., 2001).

[0180] The changes in the size of the dominant follicle, the largest subordinate follicle, and the CL, as well as the number of follicles in the size categories of 3 - 5 mm, 6 - 8 mm, and 9+ mm, were recorded once a day from the first ovulation to the first ovulation after treatment. The inter-wave interval was determined based on the period between the appearance of consecutive follicular waves.

[0181] Blood samples were also collected every 12 hours by jugular venipuncture to measure the plasma LH concentration from - 36 hours to +72 hours of treatment. Blood samples were collected once a day to measure the plasma progesterone concentration from 1 day before treatment to 7 days after treatment. Proc Mixed repeated measures analysis of variance (SAS version 9.4) was performed to compare a series of data (follicle size, number of follicles, CL size, plasma LH, plasma progesterone).

[0182] Results: The dominant follicle stopped growing in the groups on days 1 - 2 and days 3 - 4 after cetrorelix treatment (Figure 5). The size of the dominant follicle in the untreated control group indicated that the follicle entered the quiescent phase by days 6 - 7, that is, no further increase in diameter occurred after days 6 - 7. Therefore, no difference in the diameter of the dominant follicle was expected between the heifers in the untreated control group and the heifers treated with cetrorelix on days 6 - 7.

[0183]

Table 8

[0184] There was a significant difference in the diameter of the dominant follicle between the control group and the groups on days 1 - 2 and days 3 - 4 (Table 8). This indicated that cetrorelix caused early regression of the follicles. There was no difference between the control and days 6 - 7, because the dominant follicle was already prepared to start regression.

[0185]

Table 9

[0186] The data in Table 9 showed that the waves occurred 5.3 ± 0.3 days after the first cetrorelix treatment, regardless of the stage of the dominant follicle. These results indicated a high level of synchronization of wave occurrence among the groups.

[0187] [Table 10]

[0188] The group on Days 1 - 2 had a significantly shorter wave interval. There was no difference in the ovulatory interval or the length of Wave 2. This supported that cetrorelix had a short half - life and did not continue to affect subsequent waves.

[0189] [Table 11]

[0190] The maximum CL diameter in the group on Days 1 - 2 was smaller than that in the control group. One animal in this group had a short - lived CL. The time to the maximum CL diameter was not affected by the cetrorelix treatment.

[0191] Overall, the following conclusions were obtained from this example: i. Cetrorelix caused regression of the dominant follicle regardless of the growth stage ii. New waves appeared 5.3 ± 0.3 days after the first cetrorelix injection. There was a high level of synchrony. iii. The next wave, ovulation, and cycle length were not affected. iv. The dominant follicle of the next wave became ovulable within 9 days.

[0192] (Example 5) Single injection of cetrorelix induces new follicular waves in heifers. Objective 1: To test whether a single i / m injection of 3 mg of cetrorelix into heifers on a later day after the appearance of the follicular wave (i.e., at the time of different dominant follicle states) induces the appearance of synchronous waves.

[0193] Hypothesis: 1) A single injection of cetrorelix causes regression of the dominant follicle. A new wave appears simultaneously. 2) High levels of ovulation synchrony are obtained after PGF treatment. 3) The pre-ovulatory follicles of the induced wave are fertile.

[0194] Method: A schematic diagram of the experimental design of Example 5 is shown in Figure 6.

[0195] Heifers on random days of the cycle were treated with two i / m injections of prostaglandin analog and observed for the day of ovulation (defined as day 0 of the wave) by transrectal ultrasonography. The heifers were then randomly divided into four groups (n = 7 to 8 per group) and treated with normal saline (control group) or a single i / m injection of 3 mg of cetrorelix on day 1, day 3, or day 6 of the first follicular wave. Nine days after treatment with saline or cetrorelix, the heifers were injected with a prostaglandin analog, and artificial insemination was performed when the heifers showed estrus behavior (detected by tail painting). Pregnancy diagnosis was performed 30 days after artificial insemination.

[0196] Results: The dominant follicle during the treated wave showed a growth pattern similar to that treated in Example 4. The dominant follicle began to regress 2 days after cetrorelix treatment (Figure 7, left panel). The growth pattern of the dominant follicle in the next (second) wave (Figure 7, right panel) was not affected by the previous treatment.

[0197] [Table 12]

[0198] [Table 13]

[0199]

Table 14

[0200] The following conclusions were obtained from this example: i. A new wave appeared 3.5 ± 0.4 days after a single i / m treatment with 3 mg of serotorelix. There was no effect on the state of the dominant follicle and the follicular wave with respect to the interval between treatment and wave appearance. ii. There was a high level of synchrony in both wave appearance and ovulation. iii. Oocytes produced in the second (induced) wave were fertile.

[0201] (Example 6) Effect of Serotorelix in Heifers in the Luteal Phase (High Progesterone) and the Preovulatory Phase (Low Progesterone) Specific objective: To compare the growth pattern and ovulatory capacity of the dominant follicle in beef heifers after treatment with serotorelix in the luteal phase (days 5 - 6 of the wave during the period of a functional corpus luteum) and the preovulatory phase (days 5 - 6 of the wave during the period of a regressing corpus luteum).

[0202] Hypothesis: 1. Serotorelix regresses both the dominant follicle in mid - diestrus and the dominant follicle in the pre - ovulatory stage, and 2. The dominant follicle in animals treated with serotorelix does not ovulate in response to exogenous LH (hCG).

[0203] Experimental design: The study was conducted in pubertal Hereford crossbred beef heifers (n = 24) at the Livestock and Forage Centre of Excellence (LFCE) of the University of Saskatchewan using a 2×2 factorial design (Table 15). An initial transrectal ultrasound examination was performed to confirm post - pubertal or post - partum cyclicity by the presence or absence of a corpus luteum (CL). Animals with a CL were weighed, treated with a prostaglandin F2α analogue as in Example 4, and monitored by ultrasound to detect ovulation. The ovulation day was considered the day on which the wave appeared (defined as day 0).

[0204]

Table 15

[0205] On the day of ovulation, heifers were randomly divided into four groups. Prostaglandin was administered to the heifers in the pre-ovulatory group on Days 4 and 4.5 of the wave, while the corpus luteum (CL) was maintained in the heifers in the luteal group (i.e., no prostaglandin was administered). The treatments were carried out as outlined in Table 16 and Figure 8 of the schematic schedule. The heifers in the cetrorelix group were administered 3 mg of 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 hours after the cetrorelix / saline treatment) to determine the ovulatory capacity of the dominant follicle.

[0206]

Table 16

[0207] Blood samples were collected 24 hours after cetrorelix or no treatment (control group) to measure the plasma concentrations of LH and progesterone. The data were analyzed in the same manner as in Example 4.

[0208] Results: Ovulation occurred in all four groups after treatment with hCG (cetrorelix and control, low and high progesterone), which, in contrast to the proposed hypothesis 2, indicates that the dominant follicles in the high progesterone environment and the pre-ovulatory follicles in the low progesterone environment maintained their ovulatory capacity for at least 48 hours after cetrorelix treatment and had not regressed yet, i.e., the follicles ovulated in response to exogenous hCG (LH). The heifers in the cetrorelix group ovulated on Day 9 or 10 (3 to 4 days after the cetrorelix treatment), and a new follicle wave began. This period is consistent with previous experiments indicating that the cetrorelix treatment and subsequent LH or hCG can also be an effective means to initiate the appearance of synchronized follicle waves.

[0209] (Example 7) Effect of a single injection of cetrorelix on random days of the follicular wave (and estrous cycle) in heifers First objective: To investigate the effect of a cetrorelix treatment performed on random days of the cycle on follicular wave synchronization, ovulation synchronization, and pregnancy rate after timed artificial insemination in heifers.

[0210] Hypotheses: 1) The cetrorelix treatment performed on random days of the cycle simultaneously induces a new wave, 2) The cetrorelix protocol results in ovulation synchronization and an acceptable pregnancy rate after timed artificial insemination, and 3) This steroid-free ovulation synchronization protocol is as effective as the estradiol-based timed AI protocol.

[0211] Method: A schematic diagram of the experimental design of Example 7 is shown in Figure 9.

[0212] This experiment was conducted in 38 mature Angus heifers from the Federal University of Santa Maria in Brazil. Heifers on random days of the cycle (defined as day 0) were divided into two 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 heifers at this time. Eight days after the cetrorelix or estradiol treatment, the heifers were given an i / m injection of 500 μg of cloprostenol (a prostaglandin analogue), and the CIDR device was removed. Artificial insemination (AI) was performed when the heifers showed estrus behavior (detected by tail painting). At the time of artificial insemination, the heifers were given an i / m injection of 250 μg of gonadorelin acetate (a GnRH analogue). If ovulation did not occur within 24 hours of the GnRH treatment, a second AI was performed. Pregnancy diagnosis was performed 29 - 30 days after artificial insemination.

[0213] Results: A higher proportion of heifers showed estrous behavior after cetrorelix treatment than after estradiol - progesterone treatment (Figure 10, left panel). In both treatment groups, the majority of ovulations occurred between 72 and 96 hours after CIDR removal (Figure 10, right panel).

[0214]

Table 17

[0215] The cetrorelix protocol resulted in synchronized ovulation and numerically higher pregnancy rates (79%) compared to the pregnancy rate (68%) after applying the existing standard estradiol - progesterone protocol in Angus heifers after timed artificial insemination. However, there was no statistical difference between the two treatments. The pregnancy rate did not differ between heifers inseminated once and those that required a second insemination.

[0216] The conclusions of this study are as follows: i. Based on ovulation and pregnancy data, treatment with the GnRH antagonist cetrorelix regressed the dominant follicle regardless of the follicular wave status. ii. Based on ovulation and pregnancy data, cetrorelix treatment resulted in the emergence of a new follicular wave at the same interval from the start of treatment. iii. The majority (90%) of ovulations occurred between 72 and 96 hours after prostaglandin injection in the cetrorelix protocol, and heifers showed improved estrous behavior after the cetrorelix protocol compared to the estradiol - progesterone - based protocol. iv. The pregnancy rate (79%) after the cetrorelix protocol in beef heifers was slightly better than the pregnancy rate (68%) after the estradiol - progesterone protocol, but there was no statistical difference.

[0217] (Example 8) Field trial to determine the conception (pregnancy) rate after single injection of cetrorelix on random days of the follicular wave and estrous cycle in postpartum beef cows Objective: The objective of this study was to compare the conception rates after single timed artificial insemination in multiparous cows after a cetrorelix synchronization protocol and an estradiol synchronization protocol.

[0218] Method: A schematic diagram of the experimental design of Example 8 is shown in FIG. 11.

[0219] This field trial was conducted in December 2022 at the beef cattle farm of the applicant in Brazil by the faculty of the Federal University of Santa Maria. A total of 216 postpartum beef cows of Brahman×Angus crossbreed were used in this experiment. The cows were divided into two treatment groups (n = 106 per group), and 3 mg of cetrorelix was administered i / m or estradiol benzoate on random days of the cycle (defined as day 0). A vaginal progesterone device (CIDR) was placed in all cows on day 0. The CIDR device was removed on day 8, and the animals were administered a prostaglandin F2α analogue and equine chorionic gonadotropin (eCG). Timed artificial insemination was performed 54 hours after CIDR removal, and GnRH was administered to the animals at this time. Pregnancy diagnosis was performed 30 days after artificial insemination. Transrectal ultrasonography was performed on days 0, 8, 10, and 40.

[0220] Results: The results of this study are shown in Table 18 and Table 19.

[0221] [Table 18]

[0222] The dominant follicle was larger in the cetrorelix group than in the estradiol group on the day of prostaglandin treatment (the day of CIDR removal) and the day of timed artificial insemination. The cows had a greater amount of mucus discharge and showed more estrus behavior in the cetrorelix group than in the estradiol group.

[0223]

Table 19

[0224] There was no difference in the conception (pregnancy) rate between the cetrorelix protocol (42.6%) and the estradiol-based protocol (45.6%). It should be noted that the ambient temperature exceeded 40 °C on the day of fixed-time artificial insemination, resulting in extreme heat stress, which may have affected the overall pregnancy rate in both groups. Nevertheless, there was no difference in the pregnancy rate between the two groups, indicating that the cetrorelix protocol is effective as an estradiol protocol even under extreme heat stress conditions.

[0225] Conclusion: The overall conclusion of this study is as follows: i. The preovulatory dominant follicles in the cetrorelix group were larger than those in the estradiol-based protocol on the day of CIDR removal and the day of fixed-time artificial insemination. ii. When comparing the pregnancy rates, the cetrorelix protocol was as effective as the estradiol-based protocol (i.e., there was no difference in the conception rate between the cetrorelix-based protocol and the estradiol-based protocol). The female cows showed better estrus behavior in the cetrorelix protocol.

[0226] (Example 9) Effect of Four GnRH Antagonists on LH Secretion of Bovinae Pituitary Cells In Vitro Objective: To compare the effectiveness of four GnRH antagonists (cetrorelix, degarelix, abarelix, and relugolix) on the block of GnRH-mediated LH release from gonadotropin-secreting cells of Bovinae using in vitro culture of Bovinae pituitary glands.

[0227] Hypothesis: Pituitary cells treated with GnRH antagonist in vitro have lower LH secretion levels than the untreated control group after the cells are stimulated with GnRH.

[0228] Method: Heads of mature non-pregnant female cattle (n = 4) were collected from a local meat processing plant, and the pituitary glands were dissected within 60 minutes of slaughter. Immediately after dissection, the pituitary glands were submerged in ice-cold recovery medium. The pituitary glands were washed three times in the recovery medium, and the neurohypophysis was dissected from the adenohypophysis. Using a surgical scalpel, the adenohypophysis (anterior pituitary) was sliced into 1 mm × 1 mm sections and transferred to a 50 mL Falcon tube containing DMEM medium with 0.5% BSA. The tissue sections were washed by hand-stirring the tube for 1 minute. The tissue sections were allowed to settle to the bottom of the tube, and then the supernatant was discarded. The softened sections were transferred to a 15 mL 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 with 10% SFB, and then the tube was stirred by hand for 1 minute. The dissociated cells were placed on 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 them in a DMEM solution with erythrocyte lysis buffer and then centrifuged at 200×g for 10 minutes. Then, the cells were washed with a DMEM solution and centrifuged at 200×g for 10 minutes. After the last centrifugation, the cells were resuspended in DMEM, the cell number was determined using a trypan blue assay, and the viability was evaluated. DMEM with 10% fetal bovine serum (FBS) from bovine subfamily animals was treated with 10% charcoal-dextran and passed through a 0.2 μm filter. The cells were then seeded in a 24-well tissue culture plate at a concentration of 1×10 6 viable cells per well and incubated at 37°C for 24 hours. The next day, to starve the cells, the culture medium was replaced with DMEM without FBS and left incubated for an additional 24 hours.

[0229] Treatment: Based on the results of the pilot study, the selected cetrorelix (cetrorelix acetate) concentration was 300 μg. Cetrorelix was dissolved in a small amount of DMSO and then 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 in further experiments were 300 μg for cetrorelix, 340 μg for degarelix (degarelix acetate), and 300 μg for abarelix (abarelix acetate). Both degarelix and abarelix were dissolved in ultrapure water. The equivalent amount of relugolix nanomolar concentration was 130 μg, which was dissolved in methanol. Treated cells were pre-incubated with either cetrorelix, degarelix, relugolix, or abarelix for 60 minutes. After pre-incubation, the cells were incubated with 1 nM or 10 nM GnRH (gonadorelin, Fertagyl, Merck Animal Health) for 30 minutes. The GnRH dosage was based on the pituitary research by Paolicchi et al. in 1999. The medium was collected and centrifuged at 200×g for 10 minutes to remove the cells. The positive (control) group was treated only with 1 nM and 10 nM GnRH to determine the maximum LH secretion. The LH concentration in the culture medium was measured by a commercially available ELISA kit for bovine subfamily animals and reported as mIU per 1 ml of medium.

[0230] Results: In the dissociation procedure, 20 - 50×10 6 cells per pituitary (n = 4) were obtained and the cell viability was >80%. Of note, cetrorelix is a rapid-acting peptide, relugolix is a rapid-acting non-peptide, and degarelix is a slow-acting peptide. The results of this study are explained in Figures 12 and 13 and shown in Table 20 and Table 21.

[0231]

Table 20

[0232]

Table 21

[0233] A decrease in LH secretion was predicted compared to the positive control. Cetrorelix was most effective in blocking GnRH-induced LH secretion with both 1 nM GnRH stimulation and 10 nM GnRH stimulation, i.e., the cetrorelix groups in FIGS. 12 and 13 and Tables 20 and 21 had the lowest LH levels at each GnRH concentration administered. Both degarelix and leuprorelin showed some LH suppression since they both had lower LH levels than the control. At the concentrations used, abarelix did not affect LH suppression with 1 nM GnRH exposure but slightly decreased LH concentration when exposed to 10 nM LH. When comparing the results of 1 nM and 10 nM GnRH exposure among the same drugs, an increase in the dose-dependence of LH secretion was recorded. Furthermore, the ranking of the drugs remained invariant between 1 nM GnRH exposure and 10 nM GnRH exposure.

[0234] Conclusion: The overall conclusion of the study is as follows: i. Cetrorelix is an immediate-acting GnRH antagonist peptide, leuprorelin is an immediate-acting non-peptide, and degarelix is a delayed-acting peptide. These three GnRH antagonists blocked GnRH-induced LH secretion compared to the control with low (1 nM) and high (10 nM) doses of GnRH in in vitro incubations. ii. Abarelix was unable to block LH secretion with 1 nM GnRH exposure but showed some slight blocking effect with 10 nM GnRH exposure. Abarelix is likely to require higher nanomolar concentrations to be effective at reducing LH levels. iii. Cetrorelix was the most effective antagonist at reducing LH levels. iv. There were differences in the blocking efficiency of GnRH antagonists. The similar rankings of cetrorelix, degarelix, relugolix, and abarelix at 1 nM and 10 nM GnRH exposure indicate the consistency of the responses. v. The results of this experiment support that the mechanism underlying the action (i.e., the ability to block LH secretion from the pituitary gland) is similar among the four tested GnRH antagonists, but their blocking efficiencies at certain nanomolar concentrations differed among the drugs. Therefore, based on these results, degarelix, relugolix, and abarelix, as well as other GnRH antagonists, are predicted to have in vivo effects similar to those shown by cetrorelix, for example, on the synchronization of the emergence of follicular waves, but calibration of the effective dosage will be required for each drug.

[0235] (Example 10) Effect of four GnRH antagonists (cetrorelix, degarelix, relugolix, abarelix) on GnRH-induced plasma LH levels and ovulation in beef cattle Objective: To identify the effect of GnRH antagonists on plasma LH levels and ovulation in heifers after GnRH injection.

[0236] Hypothesis: 1) GnRH antagonists (cetrorelix, degarelix, relugolix, abarelix) prevent the increase in LH regardless of GnRH administration. 2) Animals treated with GnRH antagonists do not ovulate.

[0237] Method: Pubertal heifers (n = 10) with functional CL from the Federal University of Santa Maria were selected. The heifers were given an intramuscular (i / m) injection of prostaglandin F2α analog (PGF, Lutalyse 5 ml or Estamate 2 ml) to induce CL regression. The heifers were randomly divided into four treatment groups at the time of PGF injection: Trt1 (n = 2, 3 mg of abarelix), Trt2 (n = 2, 3 mg of degarelix), Trt3 (n = 2, 3 mg of relugolix), and Trt4 (n = 4, 3 mg of cetrorelix). The heifers were treated with these GnRH antagonists 1.5 days (36 hours) after PGF injection, and the time point of GnRH antagonist treatment was defined as -60 minutes. The heifers were treated with GnRH (2 mg of Fertagyl i / m) at 0 minutes to induce ovulation. Jugular venous puncture plasma samples were collected every 60 minutes for 3 hours (-60 minutes to 120 minutes; with the time of GnRH administration as t = 0 minutes). The plasma samples were maintained at 4°C overnight and LH analysis was performed the next day using a commercially available ELISA kit for bovine subfamily animals. Transrectal ultrasonography was performed at the time of GnRH injection (time point 0 hours) and also 2 days later (48 hours after GnRH injection) to record the size of the dominant follicle after treatment and detect ovulation.

[0238] Results: Figure 14A shows an example of proof of concept using cetrorelix. GnRH injection was performed at 0 minutes: The untreated control showed a 1.5-fold increase in plasma LH concentration within 30 minutes, while LH levels remained unchanged in heifers pretreated with cetrorelix 60 minutes before GnRH injection. Table 22 shows the plasma LH concentrations of individual animals at -60 hours (time point of GnRH antagonist injection), 0 minutes (GnRH exposure), and at 60 minutes and 120 minutes after GnRH injection.

[0239]

Table 22

[0240] In Table 22 (Table 22), since there were large individual variations in plasma LH concentrations before treatment with the LH antagonist, the data were normalized against the time point of GnRH injection (considered as 1), and the fold change for each group is shown in Figure 14B. In the positive control group (i.e., no GnRH antagonist treatment was performed), the LH concentration increased 1.57-fold in 2 hours. On the other hand, animals pretreated with abarelix, degarelix, leuprorelin, or cetrorelix did not show an increase in LH when exposed to GnRH, indicating that all four GnRH antagonist drugs tested were effective in blocking GnRH-induced LH secretion in vivo. Table 23 (Table 23) shows the size of the dominant follicle at the time of GnRH treatment and 48 hours later, and whether ovulation was detected up to 48 hours after GnRH treatment. All beef cattle pretreated with any of abarelix, degarelix, leuprorelin, or cetrorelix were unable to ovulate by 48 hours after GnRH injection, while both positive control group animals (i.e., animals administered only GnRH) ovulated by 48 hours.

[0241]

Table 23

[0242] Conclusion: Both hypotheses are supported. All four GnRH antagonists (abarelix, degarelix, leuprorelin, and cetrorelix) blocked GnRH-induced LH release and prevented GnRH-induced ovulation in beef cattle, indicating that any of these GnRH antagonist drugs can be used in the breeding management protocol described herein.

[0243] (Example 11) Effect of GnRH Antagonist on Follicular Dynamics in Female Cattle Objective: To test the effect of administration of other GnRH antagonists (selected drugs including, but not limited to, degarelix, abarelix, ganirelix, antide, relugolix, elagolix, or asrelin) on follicular wave emergence when injected on Days 1 and 2, 3 and 4, or 5 and 6 after follicular wave emergence.

[0244] Method: The schematic diagram of the experimental design of Example 11 is shown in Figure 15.

[0245] The experimental design is very similar to Example 4, except that PGF is used to induce ovulation before the start of the experiment, and FA (follicular ablation) is performed 7 days after PGF (approximately 3 - 4 days after ovulation).

[0246] Treatment with two i / m injections (24 - hour interval) is used. One or more of the following drugs are tested in parallel: degarelix, abarelix, ganirelix, antide, relugolix, elagolix, or asrelin. The treatment is performed on Days 1 - 2, or 3 - 4, or 5 - 6 of the wave.

[0247] For animal reuse or breeding (i.e., not part of the experimental design), PGF is administered on Day 4 of the emergence of a new (post - treatment) wave to induce ovulation.

[0248] The following endpoints are measured: - Measurement of the diameter of the largest (dominant) follicle present at the time of administration of the treatment once a day (until regression or ovulation) - The day on which a new wave appears after treatment - Measurement of the diameter of the dominant follicle of the new wave after treatment once a day - The day on which ovulation occurs after treatment - The day on which ovulation occurs after wave emergence - Diameter of the CL once a day - Doppler echo images of the CL (visual scoring from grades 1 to 4): Grade 1 = functional diestrus CL at the time of PGF injection, Grade 2 = 24 hours after PGF, Grade 3 = 48 - 72 hours after PGF, Grade 4 = 1 day after ovulation detection (late estrus). The diameter of the CL is also used for the grading scheme. - For progesterone, blood samples 48 hours after the end of GnRH antagonist treatment

[0249] Expected results: The appearance of follicular waves is synchronized by the GnRH antagonists tested.

[0250] (Example 12) Comparison of various GnRH antagonists Objective: To compare the effects of various GnRH antagonists on follicular dynamics in dairy cows

[0251] The effects of other GnRH antagonists (e.g., asilin, antarelix / teverelix, degarelix, ganirelix, antide, relugolix, elagolix, abarelix, plazarelix, lamorelix, antide, detirelix, ozerelix, linzagolix, opigolix, sufgolix, and / or A - 75998) are tested in parallel and compared with cetrorelix.

[0252] Method: A schematic diagram of the experimental design of Example 12 is shown in Figure 16. The experimental design and endpoints are very similar to Example 11, except that only one administration time point (days 3 - 4 of the wave) for drug treatment is tested.

[0253] Expected results: Cetrorelix, asilin, antarelix / teverelix, degarelix, ganirelix, antide, relugolix, elagolix, abarelix, plazarelix, lamorelix, antide, detirelix, ozerelix, linzagolix, opigolix, sufgolix, and / or A - 75998 all synchronize the appearance of follicular waves, but the period between treatment and the day of FWE may vary among the drugs tested.

[0254] (Example 13) Comparison between single dose and double dose Objective: To compare the effects of single injection and double injection (24-hour interval) of treatments (cetrorelix and degarelix / other GnRH antagonists) on follicular dynamics in heifers.

[0255] Method: A schematic diagram of the experimental design of Example 13 is shown in Figure 17. The experimental design and endpoints are very similar to those of Example 11, except that the number of administrations of GnRH antagonist (1 time and 2 times) administered on the 3rd day or the 3rd - 4th day is compared respectively.

[0256] Expected result: Administration of a single dose or a double dose series of GnRH antagonist synchronizes the FWE, but the period between the treatment and the day of FWE may vary.

[0257] (Example 14) Optimization of cetrorelix dosage in beef cows Objective: To determine the minimum effective i / m dose of a single injection of cetrorelix for synchronizing wave emergence and ovulation.

[0258] Method: A schematic diagram of the experimental design of Example 14 is shown in Figure 18. Lactating beef cows on random days of the cycle (defined as day 0) were divided into three groups (n = 15 per group) and treated with a single i / m injection of 1) 3 mg of cetrorelix, 2) 2 mg of cetrorelix, or 3) 1 mg of cetrorelix. A progesterone-releasing device (CIDR) was placed in the vagina of the heifer at this time. Eight days after the cetrorelix treatment, the cows were injected with a prostaglandin analogue and the CIDR device was removed. At the time of CIDR removal, the animals were tail-painted or an estrotect patch was applied and checked once a day until ovulation. Fixed-time artificial insemination was performed 54 hours after CIDR removal and the animals were administered GnRH at this time. Pregnancy diagnosis was performed 30 days and 60 days after artificial insemination.

[0259] Measure the following endpoints: - The period between cetrorelix injection and wave appearance - Synchronization of wave appearance - Diameter of the dominant follicle at the time of PGF and AI - The period between cetrorelix and ovulation - Ovulation rate and synchronization of ovulation - Corpus luteum volume in blood flow and plasma progesterone on the 7th day after ovulation - Plasma progesterone levels on the day of cetrorelix treatment, PGF, and AI - Conception rates on the 30th and 60th days after AI

[0260] Expected result: The minimum effective i / m dose of cetrorelix is between 1 mg and 2 mg.

[0261] Although the present application has been described with reference to what is presently considered to be the preferred embodiments, it is to be understood that the application is not limited to the disclosed embodiments. On the contrary, the present application is intended to cover various modifications and equivalent configurations included within the spirit and scope of the appended claims. The claims are not to be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the entire description.

[0262] All publications, patents, and patent applications are hereby 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.

[0263] [Table 24A]

[0264] [Table 24B]

[0265] [Table 24C]

[0266]

Table 24D

[0267] Array number 1 (Human kistrin-10) YNWNSFGLRF Array number 2 (Cetrorelix-synthetic peptide) X1X2X3SYX4LRPX5 In the sequence, 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 (References) TIFF2025523198000029.tif249170TIFF2025523198000030.tif249170TIFF2025523198000031.tif250170TIFF2025523198000032.tif249170TIFF2025523198000033.tif170170

Claims

**Claim 1** A non-therapeutic method for synchronizing the appearance of follicular waves (FWE) in a female mammalian population, the method comprising administering an effective amount of a GnRH antagonist to each mammalian in the mammalian population. **Claim 2** A non-therapeutic method for synchronizing ovulation in a female mammalian population, I) a) administering an effective amount of a GnRH antagonist, and b) at a fixed time after administration of the GnRH antagonist, administering an effective amount of an ovulation inducer to each mammalian in the female mammalian population, or II) a) administering an effective amount of a GnRH antagonist and an effective amount of progesterone or a progesterone analog, b) at a fixed time after administration of the GnRH antagonist, discontinuing administration of progesterone or a progesterone analog and administering an effective amount of prostaglandin (PGF) or a PGF analog, and c) at a fixed time after discontinuing progesterone or a progesterone analog and / or after administering PGF or a PGF analog, administering an effective amount of an ovulation inducer to each mammalian in the female mammalian population comprising the method. **Claim 3** The method according to claim 1 or claim 2, wherein the GnRH antagonist comprises cetrorelix, asrelin, antarelix / teverelix, degarelix, ganirelix, antide, leuprorelin, elagolix, abarelix, plazarelix, lamorelix, antide, detirelix, ozelrelix, linzagolix, opigolix, sufgolix, and A-75998. **Claim 4** The method according to claim 3, wherein the GnRH antagonist comprises cetrorelix. **Claim 5** The method according to claim 3, wherein the GnRH antagonist comprises abarelix. **Claim 6** The method according to claim 3, wherein the GnRH antagonist comprises degarelix. **Claim 7** The method according to claim 3, wherein the GnRH antagonist comprises leuprorelin. **Claim 8** The method according to any one of claims 1 to 7, wherein the GnRH antagonist is administered by intramuscular injection or subcutaneous injection. **Claim 9** The method according to any one of claims 1 to 8, wherein the administration of the GnRH antagonist comprises at least one treatment, and in some cases two or three treatments. **Claim 10** The method according to any one of claims 1 to 9, wherein the GnRH antagonist comprises cetrorelix delivered by intramuscular injection at a dose of about 5 μg per kg of body weight to about 200 μg per kg of body weight, optionally at a dose of about 20 μg per kg of body weight or about 40 μg per kg of body weight.

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

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

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

14. The method according to any one of claims 1 to 12, wherein the mammal is a wild animal, and optionally, the wild animal is a bison, wapiti, caribou, deer, yak, non-human primate, canine, or feline, and optionally, a tiger, lion, or leopard.

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 the step of periodically performing artificial insemination on each mammal in the female mammal population after administration of the GnRH antagonist, and optionally, the step of periodically recovering one or more embryos from each mammal in the female mammal population after artificial insemination.

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

18. The method according to any one of claims 1 to 15, further comprising the step of periodically transplanting embryos into each animal in the female mammal population after administration of the GnRH antagonist.

19. A non-therapeutic method for inducing the appearance of follicular waves (FWE) in a female mammal, the method comprising the step of administering an effective amount of a GnRH antagonist to the female mammal.

20. The method according to claim 19, wherein the GnRH antagonist comprises cetrorelix, abarelix, degarelix, ganirelix, antarelix / teverelix, plazarelix, lamorelix, antide, detirelix, ocrelizumab, asiril, elagolix, linzagolix, relugolix, opigolix, sufgolix, or A-75998.

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

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

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

24. The method according to 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 the administration of the GnRH antagonist comprises at least one treatment, and in some cases two or three treatments.

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

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

29. A non-therapeutic method for inducing ovulation in female mammals, comprising: I) a) inducing FWE as defined in any one of claims 19 to 28, and b) administering an effective amount of an ovulation inducer at regular intervals after administration of the GnRH antagonist, or II) a) inducing FWE as defined in any one of claims 19 to 28, b) co-administering an effective amount of progesterone or a progesterone analogue with the GnRH antagonist, c) stopping progesterone or a progesterone analogue and administering an effective amount of prostaglandin (PGF) or a PGF analogue at regular intervals after administration of the GnRH antagonist, and d) administering an effective amount of an ovulation inducer at regular intervals after stopping progesterone or a progesterone analogue and / or after administering PGF or a PGF analogue. A method comprising.

30. A non-therapeutic timed artificial insemination method, comprising: a) a step of inducing ovulation as defined in claim 29; and b) a step of artificially inseminating a mammal at a fixed time after administration of an ovulation-inducing agent, and optionally further comprising, c) a step of recovering one or more embryos at a fixed time after artificial insemination.

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

32. A non-therapeutic timed embryo transfer method, comprising: a) a step of inducing FWE according to the method described in any one of claims 19 to 28, or a step of inducing ovulation according to the method described in claim 29; and b) a step of transplanting one or more embryos into a female mammal at a fixed time after administration of an ovulation-inducing agent.

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

34. The method according to claim 33, further comprising, in I) c), a step of administering an effective amount of an ovulation-inducing agent at a fixed time after administration of FSH or an FSH agonist, or in II) e), a step of 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.

35. A non-therapeutic timed oocyte retrieval method, comprising: a) inducing FWE according to the method described in any one of claims 19 to 28, or inducing ovulation as defined in claims 23, 27, or 28, and b) after administration of a GnRH antagonist as defined in any one of claims 19 to 28, FSH as defined in claim 33I), PGF as defined in claim 33II), or an ovulation inducer as defined in claim 34, retrieving one or more oocytes at a fixed time.

36. I) d) After administration of an ovulation inducer, at a fixed time, artificially inseminating a female mammal, and e) after artificial insemination, at a fixed time, retrieving one or more embryos, or II) f) After administration of an ovulation inducer, at a fixed time, artificially inseminating a female mammal, and g) after artificial insemination, at a fixed time, retrieving one or more embryos, further comprising the method according to claim 34 for timed superovulation or superovulation method.

37. I) d) After administration of an ovulation inducer, at a fixed time, mating a female mammal, and e) after breeding, at a fixed time, retrieving one or more embryos, or II) f) After administration of an ovulation inducer, at a fixed time, mating a female mammal, and g) after mating, at a fixed time, retrieving one or more embryos, further comprising the method according to claim 35 or 36 for timed superovulation or superovulation method.

38. The method according to any one of claims 29 to 37, wherein PGF or a PGF analog contains PGF2a.

39. The method according to any one of claims 29 to 38, wherein the ovulation inducer contains 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 according to any one of claims 19 to 39, wherein the mammal is a breeding livestock animal, and in some cases, the breeding livestock animal is a bovine animal, a camelid animal, a horse, a pig, a sheep, or a goat.

41. The method according to any one of claims 19 to 39, wherein the mammal is a wild animal, and in some cases, a bison, a wapiti, a caribou, a deer, a yak, a non-human primate, a dog, or a cat.

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

43. The method according to any one of claims 29 to 42, wherein progesterone or a progesterone analogue is administered by a vaginal drug delivery device.

44. The method according to claim 43, wherein the mammal is a bovine subfamily animal.

45. The method according to claim 44, wherein the progesterone or progesterone analogue drug delivery device is administered for 7 to 8 days.

46. The method according to claim 45, wherein the GnRH antagonist is administered on day 0 (D0), the method comprising: i) inserting a progesterone or progesterone analogue drug delivery device on D0; ii) administering an effective amount of prostaglandin (PGF2α) or a prostaglandin analogue on day 8 (D8) and removing the progesterone or progesterone analogue drug delivery device; and iii) administering an effective amount of GnRH on day 10 (D10).

47. A drug delivery device comprising a GnRH antagonist.

48. The drug delivery device according to claim 47, which provides controlled release of the GnRH antagonist over a period of about 1 day to about 2 days.

49. The drug delivery device according to claim 47 or 48, wherein the GnRH antagonist is cetrorelix.

50. The drug delivery device according to any one of claims 47 to 49, further comprising progesterone or a progesterone analogue, and providing controlled release of progesterone or a progesterone analogue 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 a period longer than 10 days.

51. The drug delivery device according to any one of claims 47 to 50, for use in synchronizing the emergence of follicular waves (FWE) in a population of female mammals, for use in synchronizing ovulation in a population of female mammals, for use in a timed superovulation method or superovulation method, for use in a timed embryo transfer method, or for use in a timed artificial insemination method.

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

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