AU2020104306B4 - Method for enhancing assisted breeding techniques - Google Patents

Abstract

Methods are disclosed for achieving and/or enhancing assisted pregnancies (ie conception) in non human animals, particularly livestock animals such as sheep and goats. The methods are directed at predictably inducing ovulation within a relatively narrow "window" of time and involve administering to an animal in oestrus, an amount of gonadotropin releasing hormone (GnRH) or an agonist thereof which is effective to induce ovulation.

Description

METHOD FOR ENHANCING ASSISTED BREEDING TECHNIQUES TECHNICAL FIELD

[0001] The present disclosure relates to methods for achieving and/or enhancing assisted pregnancies (ie conception) in livestock animals such as sheep and goats. The methods are directed at predictably inducing ovulation within a relatively narrow "window" of time in animals in the oestrus phase.

PRIORITY DOCUMENT

[0002] The present application claims priority from Australian Provisional Patent Application No 2018903578 titled "Method for enhancing assisted breeding techniques" filed on 24 September 2018, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] The development of artificial (or "assisted") breeding techniques over the last 40 years or so has led to great increases in productivity of livestock breeding. The introduction of techniques in artificial insemination (AI), particularly laparoscopic Al, and semen collection and management has, in particular, been of enormous significance to the industry, allowing for the rapid multiplication of selected superior sire genetics and increases in pregnancy success rates within a flock/herd. In addition, the use of embryo transfer (ET) and in vitro technologies (such as in vitro fertilisation (IVF)), has achieved significant shortening in generation interval thereby accelerating genetic gain of superior maternal genetics. Further, such techniques have facilitated the importation of superior genetics or new breeds from overseas and, with increased understanding of markers and genetic mapping, it is anticipated that artificial breeding techniques such as those mentioned above will continue to play an important role in livestock breeding by assisting the controlled and/or selective multiplication and distribution of desirable traits that promise productivity and/or quality gains in a variety of areas including, for example, carcase production and qualities, fertility and product generation (eg traits affecting milk production or "wool traits") (Artificial Breeding Techniques - Improving your success rate, ProcAnn Conf ofAust White Suffolk Assoc Inc, Albury NSW, 2009).

[0004] However, despite continual refinement of artificial breeding techniques, there have been reports of declining success rates in certain livestock animals. For instance, in the Australian sheep industry, the "historical" pregnancy success rate cited for an Al program is 60-65%, but this recognises that there may be huge variation in the success rate of any one Al program of the order of 20-80%. However, it has been anecdotally reported that nowadays the typical success rate may be as low as 40-

%. While it is commonly recognised that the success of Al depends on several factors, including the technician and practices employed (Rehmana FU et al., Veterinaria 1:1-8 (2013); Inchaisri C et al., Reprod Domest Anim 46:1043-1049 (2011)), it has not yet been determined what exactly has caused this "crash" in the success rate of Al in sheep, but some thoughts include the possible reduction of semen quality, pasture effects (eg high clover populations appear to decrease fertility) and reduced drug quality, stability or animal tolerability (Sheep Artificial Breeding Company Workshop, The University Of Sydney (NSW, Australia), 28 October 2016). On the other hand, in dairy cattle, there is substantial evidence that fertility is decreasing with increasing milk yield (Royal MD et al., Animal Science 40:487-501 (2000); Butler WR., Livestock Production Science 83:211-218 (2003)). In addition, it has been reported that high milk production increases the number of silent heats (Harrison RO et al., J Dairy Sci 73:2749-2758 (1990)) and that the stresses and strains of such high milk production may disrupt hormonal equilibrium in dairy cattle, reducing oestrus intensity, and lowering luteinising hormone (LH) resulting in the failure of ovulation (Dobson H et al., Animal 2(8):1104 1111 (2008)).

[0005] Thus, in the light of observed reductions in the success rate of Aland other artificial breeding techniques, there is an ongoing need to identify and develop new methods and protocols for achieving and/or enhancing assisted pregnancies (ie conception) in livestock.

SUMMARY

[0006] According to a first aspect, the present disclosure provides a method of inducing ovulation in a group of sheep for artificial insemination (Al), wherein said method comprises the steps of: providing and/or selecting sheep in oestrus by (i) treating each sheep within the group with a controlled internal drug (progesterone) release device (CIDR) for a period of 10-14 days; and (ii) removing said CIDR from the sheep and substantially immediately thereafter administering to each sheep, by a single im or iv injection, chorionic gonadotropin (Gn) and, optionally, exposing the sheep to a teaser to induce oestrus; and administering to each sheep in oestrus, at 25-35 hours of removing said CIDR, an amount of gonadotropin releasing hormone (GnRH) or an agonist thereof which is effective to induce synchronised ovulation within a 2 hour window of time.

[0007] Sheep treated in accordance with the method of the first aspect maybe subjected to artificial insemination (Al) with thawed frozen semen.

[0008] In a second aspect, the present disclosure provides a method of artificially inseminating a sheep, wherein the method comprises inducing ovulation in a sheep in a group treated in accordance with the method of the first aspect, and subjecting the sheep to artificial insemination (AI) with thawed frozen semen, preferably by laparoscopic insemination (LapAl).

[0009] In a further aspect, the present disclosure provides a kit comprising at least a container provided with gonadotropin releasing hormone (GnRH) or an agonist thereof, optionally packaged together with instructions for use of the kit in the method of the first or second aspects.

DETAILED DESCRIPTION

[0010] The present disclosure relates to methods for achieving and/or enhancing assisted pregnancies (ie conception) in livestock animals, particularly sheep. The methods are directed at predictably inducing ovulation within a relatively narrow window of time in animals in the oestrus phase of the oestrous cycle.

[0011] In a first aspect, the present disclosure provides a method of inducing ovulation in a group of sheep for artificial insemination (AI), wherein said method comprises the steps of: providing and/or selecting sheep in oestrus by (i) treating each sheep within the group with a controlled internal drug (progesterone) release device (CIDR) for a period of 10-14 days; and (ii) removing said CIDR from the sheep and substantially immediately thereafter administering to each sheep, by a single im or iv injection, chorionic gonadotropin (Gn) and, optionally, exposing the sheep to a teaser to induce oestrus; and administering to each sheep in oestrus, at 25-35 hours of removing said CIDR, an amount of gonadotropin releasing hormone (GnRH) or an agonist thereof which is effective to induce synchronised ovulation within a 2 hour window of time.

[0012] Animals treated in accordance with the method of the first aspect may be subjected to artificial insemination (AI) with thawed frozen semen.

[0013] Typically, the method of the first aspect will be simultaneously performed on multiple animals or a flock/herd of suitable female animals (eg 5-250 animals) and, accordingly, the animals will be subjected to one or more techniques for inducing and synchronising oestrus in all of the animals. For example, the animals may be subjected to the "ram effect" where a male animal is introduced to a group of female animals unaccustomed to the presence of a male, preferably in combination with a progesterone or progestin pre-treatment (eg where a single dose of progesterone (eg 20 mg dose for sheep) is administered to each animal at, or 1-2 days prior to, introduction of the male), to synchronise oestrus. Alternatively, each animal may be treated with progesterone/progestin treatment over a sustained period such as, for example, daily administration of progesterone or melengestrol acetate for a minimum of 5 to 7 days or, more conveniently, by using a sustained progesterone release device such as a controlled internal drug (progesterone) release device (CIDR) as are well known to those skilled in the art. CIDRs are commercially available and are typically placed in the animal for a period of 5 to 24 days; generally 10 to 14 days in sheep (preferably, about 12 days) and 18 to 21 days in goats. Such a CIDR will generally deliver a dose of 250 to 350 mg of progesterone over the period that it is placed in the animal at a rate of, for example, 20 to 30 mg/day. Completion of the progesterone/progestin treatment (eg by removal of a CIDR), and the immediate administration of gonadotropin (Gn), induces the oestrus phase in the animal (typically within 24 hours (eg within 24 hours of "CIDR out") and continuing until up to about 60 hours post CIDR out).

[0014] Where intended for an Al program, animals treated with a CIDR will, following removal of the CIDR, typically be administered with a dose of a gonadotropin (Gn) (eg immediately at the time of the device removal or otherwise within 1-2 days) and, optionally, exposed to teasers (eg androgenised wethers or castrated males), such that the animals in the flock/herd are synchronised in the oestrus phase, ready for the step of administering gonadotropin releasing hormone (GnRH) or an agonist thereof. Otherwise, where intended for an embryo collection (EC) program, the animals will already be undergoing treatment to stimulate super-ovulation (eg using super-ovulatory hormone treatment), and following removal of the CIDR (ie to induce the oestrus phase), the animals are already synchronised in the oestrus phase, and accordingly, proceed to the step of administering GnRH or an agonist thereof (usually within about 1 day) typically without the need for any gonadotropin (Gn) administration or exposure to teasers.

[0015] Suitable gonadotropin (Gn) preparations for administration to sheep include, for example, equine chorionic gonadotropin (eCG) (also known as pregnant mare's serum gonadotropin (PMSG)) such as the commercially available Pregnecol@ product (Vetoquinol Australia, Brisbane Airport, QLD, Australia). Typically, the Gn preparation will be administered at a dose in the range of 400-700 iu (sheep).

[0016] Preferably, the GnRH or agonist thereof is administered in an amount effective to induce ovulation in the oestrus-phase animal within about 12 to 22 hours or, more preferably, within about 13-20 hours of the administration of the GnRH or agonist thereof (eg at about 18 hours from the time of administration of the GnRH or agonist thereof). Where the animal has been treated with progesterone as described in the preceding paragraph, then typically, the GnRH or agonist thereof will be administered within 15 to 40 hours (for animals intended for an Al program, preferably at 25 to 35 hours and, more preferably, at about 30 hours.

[0017] Thus, in some embodiments where the animal is intended for anAl program and has been treated with progesterone via a CIDR, the GnRH or agonist thereof is administered in an amount effective to induce ovulation in the oestrus-phase animal within about 45 to 55 hours, more preferably within about 45 to 50 hours, post CIDR removal. Preferably, the ovulation will occur in a majority of the animals within a treated flock/herd within about a 2 hour window of time within these periods. Further, in accordance with some preferred embodiments where the animal is intended for an Al program and has been treated with progesterone via a or CIDR, the GnRH or agonist thereof is administered in an amount effective to induce ovulation in the oestrus-phase animal within about 47 to 49 hours post CIDR removal (eg at about 48 hours from the time of CIDR removal).

[0018] The exact amount of the GnRH or agonist thereof required to be administered to the animal to induce ovulation within the time periods indicated in the preceding paragraph [0016] may, as will be readily appreciated by those skilled in the art, vary and depend upon a number of factors including, for example, the activity of the particular GnRH or GnRH agonist used, the metabolic stability and length of action of the particular GnRH or GnRH agonist, the age, body weight and/or condition of the animal, and the route and time of administration of the GnRH or agonist thereof. However, for a GnRH such as gonadorelin acetate, the amount administered to the animal may typically be in the range of about 25 to 100 g per animal. Preferably, the amount will be about 50 g per animal.

[0019] The GnRH or agonist thereof will typically be administered in a single dose, preferably by intramuscular (im) or intravenous (iv) injection.

[0020] Preferably, the method of the first aspect comprises administering gonadorelin acetate; for example, commercially available Fertagyl®(Intervet Australia Pty Limited, Bendigo East, VIC, Australia), GONAbreed@ (Parnell Pharmaceuticals Holdings Ltd, Overland Park, KS, United States of America), and OvurelinTM (Bayer AG, Leverkusen, Germany). However, those skilled in the art will readily appreciate that any of the well known range of GnRH agonists may also be suitable. Particular examples of such GnRH agonists include, for example, buserelin, deslorelin, goserelin, histrelin, leuprorelin, meterelin, nafarelin, triptorelin and combinations thereof.

[0021] Animals treated in accordance with the method of the first aspect ought to ovulate within a relatively narrow or "tight" window of time (eg a 2 hour period).

[0022] Thus, in an Al program, this provides the opportunity to more accurately match or synchronise insemination with ovulation to bring about an increased conception rate. When used in an Al program, the animals will preferably be inseminated (eg within a 2 hour window of time during which ovulation is expected) using laparoscopic insemination (LapAl) wherein an animal is restrained in a laparoscopic table and the uterus viewed via a telescope inserted through the abdominal wall. This technique advantageously enables thawed frozen semen to be placed into each uterine horn (via a sterile pipette) when the oocyte(s) is expected to be within the oviduct, thereby enhancing the likelihood of fertilisation.

[0023] Further, the treatment of animals in accordance with the method of the first aspect, so as to induce ovulation within a relatively narrow window of time (eg a 2 hour period) may also be usefully employed in a process for recipient in vivo embryo transfer (RIVET). In particular, in such a process wherein the genetics of a particularly valuable livestock animal (or even an animal of an endangered exotic species) may be preserved and/or expanded (eg where that animal is no longer able to breed) by collecting oocytes and transferring those oocytes to an intermediary recipient ("Egg Box" ) to produce multiple embryos (following insemination) for transfer to final recipient animals, the process can benefit by employing the method of the first aspect so as to induce ovulation, within a relatively narrow window of time (eg about a 2 hour window of time), ahead of the embryo transfer (ET). In such an application, the GnRH or agonist thereof is administered in an amount effective to induce ovulation in the oestrus-phase animal within about 47 to 49 hours post sponge/CIDR removal (eg at about 48 hours from the time of sponge/CIDR removal). Typically, the ET to the final recipient animals will take place between about 3 and 6 days (preferably, 5 days) following ovulation.

[0024] In a second aspect, the present disclosure provides a method of artificially inseminating a sheep, wherein the method comprises inducing ovulation in a sheep in accordance with the method of the first aspect, and subjecting the sheep to artificial insemination (AI) with thawed frozen semen, preferably by laparoscopic insemination (LapAl).

[0025] In a further aspect, the present disclosure provides a kit comprising at least a container provided with gonadotropin releasing hormone (GnRH) or an agonist thereof, optionally packaged together with instructions for use of the kit in the method of the first or second aspects.

[0026] In a still further aspect, the present disclosure provides the use of a gonadotropin releasing hormone (GnRH) or an agonist thereof to induce ovulation in a sheep, wherein said sheep is in oestrus.

[0027] Similarly, the present disclosure also provides the use of a gonadotropin releasing hormone (GnRH) or an agonist thereof in the manufacture of a medicament to induce ovulation in a sheep which is in oestrus. The embodiments of the methods of the present disclosure described above may also be applicable to these uses.

[0028] The methods of the present disclosure are hereinafter further described with reference to the following, non-limiting examples.

EXAMPLES

Example 1 Timed ovulation in laparoscopic Al program

MaterialsandMethods

[0029] Sheep (White Suffolk and White Dorper breeds) were treated according to a standard Al protocol or an enhanced Al protocol according to the present disclosure as follows:

[0030] Standard Al protocol The standard Al protocol followed a present Australian industry laparoscopic artificial insemination (LapAl) protocol. Briefly, a commercial controlled internal drug (300 mg progesterone) release device (CIDR; Eazi-BreedTM CIDR@ sheep insert; Zoetis Services LLC, Parsippany, NJ, United States of America) is first intra-vaginally placed into each ewe, and then after 12 days or cessation of progesterone release, the CIDRs are removed, and the sheep administered with 400 iu of gonadotropin (Gn; Pregnecol@) by intramuscular (im) or intravenous (iv) injection. The sheep are then exposed to 3% androgenised teasers (ie teasers are run at a minimum ratio of 3 per 100 ewes) to detect animals in oestrus; ewes marked by a teaser are drafted and inseminated laparoscopically using thawed frozen semen (usually from about 53 hours after CIDR removal). Depending on the numbers programmed on the day, ewes are inseminated up to 60 hours post-CIDR removal.

[0031] Enhanced Al protocol As with the standard Al protocol, in the enhanced Al protocol a 300 mg progesterone CIDR (eg Eazi BreedTM CIDR@ sheep insert) is intra-vaginally placed into each ewe. Following the removal of the CIDR, the sheep are administered with 400 iu of gonadotropin (Gn; Pregnecol@) by im or iv injection and exposed to 3% androgenised teasers. Then, 30 hours post-CIDR removal, the ewes are administered with 50 g gonadorelin acetate, a synthetic gonadotropin-releasing hormone (GnRH; Fertagyl@ 0.1 mg/ml). Laparoscopic insemination is then performed on each ewe at about 48 hours post-CIDR removal (ie 48 hr 1 hr) using thawed frozen semen. Accordingly, the LapAI procedure is performed in the animals in the order that the CIDRs are removed. In addition, removal of the CIDRs in accordance with the enhanced protocol is performed in animal groups (25 ewes per group) staggered at appropriate intervals (see Table 1) to enable all animals to be conveniently inseminated at the 48 hour post-CIDR removal time-point.

Results

[0032] In a 100 ewe flock of White Suffolk, the standard Al protocol achieved a conception rate of % (ie where the pregnancy was detected by standard ultrasound methodologies), while the enhanced

Al protocol of the present disclosure achieved a greatly enhanced conception rate of 95%. Similar results were achieved in a flock of White Dorper ewes; that is, the enhanced Al protocol achieved an % conception rate compared to the historical 45%-60% conception rates. In some animals, during the LapAl procedure, ovarian follicles were observed through the telescope for (over) a period of about hour (ie prior to the placement of semen into the uterus), enabling the observation of actual ovulation or otherwise enabling the accurate prediction of the timing of ovulation.

Discussion

[0033] With the standard Al protocol, recipient sheep are inseminated over a 7 hour window (ie between about 53 and 60 hours post-CIDR removal). In contrast, using the enhanced Al protocol according to the present disclosure, the sheep are predictably induced to ovulate at 48 hours post CIDR removal (plus or minus one hour) and insemination is timed to occur within a comparatively "tight" 2 hour window between about 47 and 49 hours post-CIDR removal. While not wishing to be bound by theory, the present applicant considers that the enhanced Al protocol thereby offers superior synchronicity between ovulation and insemination to bring about an increased conception rate. In other words, the enhanced Al protocol reduces the likelihood of the semen being placed in the uterus asynchronous to ovulation. In addition, the predictability of the timing of ovulation in sheep treated in accordance with the enhanced Al protocol readily enables management of the flock so that ewes are inseminated at the 48 hour time-point throughout the course of the day. Table 1 sets out the management of a program of an enhanced Al treatment protocol for 100 ewes, comprising four groups of 25 ewes. However, the group size can be readily varied according to technician speed (ie particularly, by how many inseminations the technician can perform per hour).

Table 1

Day & Time Ewe group Enhanced Al protocol step Day 0 7.00 am Groups 1-4 100 ewes CIDR in Day 12 8.00 am Group 1 25 ewes CIDR out + 400 iu Pregnecol@ + teasers 9.00 am Group 2 25 ewes CIDR out + 400 iu Pregnecol@ + teasers 11.00 am Group 3 25 ewes CIDR out + 400 iu Pregnecol@ + teasers 12.00 pm Group 4 25 ewes CIDR out + 400 iu Pregnecol@ + teasers Day 13 2.00 pm Group 1 25 ewes + 0.5 ml GnRH 3.00 pm Group 2 25 ewes + 0.5 ml GnRH

5.00 pm Group 3 25 ewes + 0.5 ml GnRH 6.00 pm Group 4 25 ewes + 0.5 ml GnRH Day 14 8.00 am Group 1 25 ewes LapAl 9.00 am Group 2 25 ewes LapAl 11.00 am Group 3 25 ewes LapAl 12.00 pm Group 4 25 ewes LapAl

Example 2 Timed ovulation in embryo collection (EC) and transfer (ET) program

Materials andMethods

[0034] Preparation of donor maiden ewes and embryo collection Maiden ewes are prepared for donor embryo collection (EC) by exposing the animals to 5% androgenised teasers for six weeks. Cycling ewes are then selected for the EC program (see Table 2 for the management of a program of an enhanced EC treatment protocol). The selected ewes are in forward store condition and on a rising plain of nutrition (eg on a dietary supplement of lupins (250 g per day) for six weeks prior to embryo collection). At the commencement of the program, a 300 mg progesterone CIDR (Eazi-BreedTMCIDR@ sheep insert) is intra-vaginally placed into each ewe. 10 days later, ewes begin treatment for super-ovulation by an initial administration of follicle stimulating hormone (FSH) (eg Folltropin@; Vetoquinol Australia). This is then followed up with further FSH administrations on Days 11 and 12 as set out in Table 2. On Day 12, the ewes are also administered with prostaglandin (PG) (eg a 1 ml dose of 250 pg/ml cloprestenol; Estrumate@, Intervet Australia) and the CIDR is removed. On Day 13, the animals are administered with further FSH and PG doses (ie the animals receive a "double dose" of PG to assist in bringing the animals into the oestrus phase), followed by (12 hours later) a 50 pg dose of gonadorelin acetate (GnRH; Fertagyl@ 0.1 mg/ml). This dose of GnRH is given at about 24 hours post-CIDR removal. The ewes are then observed to confirm that they are in the oestrus phase (ie through marking by a teaser or by showing standing heat) and drafted for laparoscopic insemination (LapA) at about 37-39 hours post-CIDR removal (ie 38 hr 1 hr). The inseminated animals are then subjected to standard embryo collection (EC) surgery on Day 19 and the embryos stored or transferred to recipient ewes.

[0035] Preparation of recipient ewes and embryo transfer (of embryo(s) from maiden donor ewes) Recipient ewes (ie ewes to receive embryo(s) by embryo transfer) are prepared "in parallel" to the donor maiden ewes. Briefly, on the same day that the maiden ewes receive a CIDR, a CIDR is intra vaginally inserted into the ewes and then on Day 12, the CIDR is removed from the recipient ewes and a 400 iu dose of gonadotropin (Gn) (Pregnecol@) administered by im or iv injection, followed by exposure to teasers. Then, 20-22 hours later, the ewes are administered with prostaglandin (PG) (eg a 0.5 ml dose of 250 pg/ml cloprestenol; Estrumate@). Transfer of an embryo(s) collected from the donor animals is then performed on the recipient ewes at Day 19 in accordance with standard ET techniques.

[0036] Preparation of donor mature ewes and embryo collection Mature ewes are prepared for donor embryo collection (EC) in a similar manner to that described for the maiden ewes above (see also Table 3). However, PG administration is not used and LapAl is conducted at about 48 hours post-CIDR removal (ie48 hr 1 hr). The step of administering GnRH (ie a 50 pg dose of gonadorelin acetate (Fertagyl@ 0.1 mg/ml) is performed at about 24 hours post-CIDR removal when the animals are expected to be in the oestrus phase. Inseminated animals are then subjected to standard embryo collection (EC) surgery on Day 19 and the embryos stored or transferred to recipient ewes.

[0037] Preparation of recipient ewes and embryo transfer (of embryo(s) from mature donor ewes) Recipient ewes (ie ewes to receive embryo(s) from the mature donor ewes by embryo transfer) are prepared in parallel to the donor mature ewes in a manner similar to that described above for maiden donor ewes (see also Table 3). Transfer of an embryo(s) collected from the donor animals is then performed on the recipient ewes at Day 19 in accordance with standard ET techniques.

Table 2 - Maiden ewe trial protocol

Day & Time Ewe group Enhanced EC protocol step Day 0 7.00 am Donors CIDR in 7.00 am Recipients CIDR in Day 10 7.00 pm Donors 3.0 mls Folltropin Day 11 7.00 am Donors 2.0 mls Folltropin 7.00 pm Donors 2.0 mils Folltropin Day 12 7.00 am Donors 1.5 mils Folltropin 6.00 pm Recipients CIDR out + 2.0 ml Pregnecol + teasers 7.00 pm Donors CIDR out + 1.5 mls Folltropin + 1.0 ml PG Day 13 7.00 am Donors 1.0 ml Folltropin + 1.0 ml PG + Dry out* 2.30 pm Recipients 0.5 ml GnRH

7.00 pm Donors 0.5 ml GnRH 7.00 pm Donors Heat detect Day 14 8.30 am Donors LapAl (37-38 hours post CIDR out) Day 18 8.00 am Donors Dry out* Day 19 8.00 am Donors EC surgery - Day 5** 12.00 pm Recipients ET

*Dry out= animal taken off food and water prior to surgery ** Day 5= 5 days post LapAl

Table 3 - Mature ewe trial protocol

Day & Time Ewe group Enhanced EC protocol step Day 0 8.00 am Donors CIDR in 8.00 am Recipients CIDR in Day 10 8.00 am Donors 3.0 mls Folltropin 5.00 pm Donors 2.0 mls Folltropin Day 11 8.00 am Donors 2.0 mls Folltropin 5.00 pm Donors 2.0 mls Folltropin Day 12 8.00 am Donors CIDR out + 2.0 mls Folltropin 8.00 am Recipients CIDR out + 2.0 ml Pregnecol 5.00 pm Donors 1.0 mls Folltropin Day 13 8.00 am Donors Dry out* 2.00 pm Donors 0.5 ml GnRH 2.00 pm Recipients 0.5 ml GnRH 5.00 pm Donors Heat detect 5.00 pm Recipients Heat detect Day 14 8.00 am Donors Heat detect

8.00 am Recipients Heat detect 8.00 am Donors LapAI (about 48 hours post CIDR out) 5.00 pm Recipients Heat detect Day 18 8.00 am Donors Dry out* 8.00 am Recipients Dry out* Day 19 8.00 am Donors EC surgery - Day 5** 12.00 pm Recipients ET

*Dry out= animal taken off food and water prior to surgery ** Day 5= 5 days post LapAl

Results and Discussion

[0038] In initial trials of 6 maiden ewes of the Merino and White Dorper breeds, the method achieved fertilisation rates above 95% with uniform, positive donor responses. Given that maiden ewes can be unpredictable/unreliable in response to super-ovulation treatment, this was an excellent result.

[0039] Subsequently, more extensive trials were performed using four groups of mature donor ewes:

Program 1 White Suffolk donor ewes (25 animals), Program 2 Merino donor ewes (14 animals), Program 3 White Suffolk and Ultra White donor ewes (16 animals), and Program 4 Texel White Suffolk donor ewes (10 animals).

The results, which are shown in Tables 4-7, indicate that very high rates of fertilisation were achieved with few unfertilised embryos (UFE; single cells that have failed to develop following insemination) being collected.

Table 4 - Program 1

Donor Ram Number of Number of Number of Number of embryos fertilised unfertilised degenerated ewe collected embryos embryos embryos transferred (UFE) (%) 15202 BG16W300 14 14(100%) 0 0

16047 BG16W300 6 6(100%) 0 0

15058 BG16W300 17 17(100%) 0 0

16058 BG16W300 6 6(100%) 0 0

15056 Warburn160048 8 7(87%) 1 0

16314 Warburn160048 10 10(100%) 0 0

15184 BG16W300 14 14(100%) 0 0

14152 BG16W300 12 12(100%) 0 0

16036 LH130572 SCOPE - I ovulation, left pregnant

16011 Warburn160048 11 11(100%) 0 0

15163 BG16W300 9 9(100%) 0 0

15115 Warburn160048 14 14(100%) 0 0

16450 Warburn160048 8 8(100%) 0 0

16102 DG160027 12 5(42%) 0 7

13291 Warburn160048 6 6(100%) 0 0

16469 LH130572 5 2(40%) 3 0

15399 LH130572 16 12(75%) 3 1

16584 LH130572 6 6(100%) 0 0

15278 BG16W300 3 3 (100%) 0 0

16532 LH130572 6 6(100%) 0 0

16228 Warburn160048 14 12(85%) 0 2

15276 Warburn160048 SCOPE -no response 15268 LH130572 SCOPE - I ovulation, left pregnant 16651 DG160027 4 4(100%) 0 0

16688 DG160027 SCOPE - I ovulation, left pregnant TOTAL 201 185(92%) 6 10

Table 5 - Program 2

Donor Ram Number of Number of Number of Number of embryos fertilised unfertilised degenerated ewe collected embryos embryos embryos transferred (UFE) (%) 150372 Gunallo 460 19 16(84%) 0 3

160228 Gunallo 460 12 10(83%) 0 2

160111 Gunallo 460 7 5(71%) 0 2

160536 Gunallo 460 3 2(66%) 0 1

160205 Gunallo 460 4 3 (75%) 0 1

160207 Gunallo 460 9 7(77%) 1 1

130401 Gunallo 460 9 1 (11%) 8 0

140319 Ric 721 16 14(77%) 0 2

140340 Ric 721 18 18(100%) 0 0

130118 Ric 721 8 8(100%) 0 0

160660 Ric 721 17 12(70%) 0 5

160558 Ric 721 4 4(100%) 0 0

160629 Gunallo 008 6 6(100%) 0 0

120010 Ric 721 18 18(100%) 0 0

TOTAL 150 124(83%) 9 17

Table 6 - Program 3

Donor Ram Number of Number of Number of Number of ewe embryos fertilised unfertilised degenerated collected embryos embryos embryos transferred (%) (UFE)

160287 A277 6 5 (83%) 1 0

150122 DG27 19 18(94%) 1 0

160170 DG27 9 8 (88%) 0 1

160811 UW714 12 12(100%) 0 0

130822 UW714 17 17(100%) 0 0

130804 UW714 16 16(100%) 0 0

160803 UW693 10 10(100%) 0 0

160873 UW693 11 11(100%) 0 0

160897 UW693 11 10(90%) 0 1

130021 S153 9 9(100%) 0 0

158023 A277 4 4(100%) 0 0

130825 UW714 18 18(100%) 0 0

140647 UW693 13 13 (100%) 0 0

150007 DG27 5 2(40%) 2 1

150336 S153 5 3 (60%) 2 0

160173 S153 5 4(80%) 1 0

TOTAL 170 160(94%) 7 3

Table 7 - Program 4

Donor Ram Number of Number of Number of Number of ewe embryos fertilised unfertilised degenerated collected embryos embryos embryos transferred (%) (UFE)

12/7038 DG160077 10 9(90%) 0 1

14/8215 DG160077 10 10(100%) 0 0

16/365 DG160077 6 6(100%) 0 0

12/7011 SB16060 13 11(85%) 2 0

/298 SB16060 3 2(66%) 0 1

14/069 98/108 8 5(63%) 2 1

14/320 SB16060 5 5(100%) 0 0

/8020 SB16060 14 12(86%) 2 0

14/7345 SB16060 3 3(100%) 0 0

12/047 SB16060 18 18(100%) 0 0

TOTAL 90 81(90%) 6 3

Example 3 Timed ovulation in for recipient in vivo embryo transfer (RIVET) program

[0040] The method of the present disclosure may be usefully employed in a process for recipient in vivo embryo transfer (RIVET). In particular, in a RIVET process intended to synchronise the oestrus cycle of a donor ewe, intermediary recipient ewes (Egg Box) and final recipient ewes, recipient ewes may be induced to ovulate, within a relatively narrow window of time (eg about a 2 hour window of time), ahead of embryo transfer (ET).

Materials andMethods

[0041] Preparation of donor ewe and oocyte collection A ewe is prepared for oocyte collection (OC) by exposing the animal to 5% androgenised teasers for six weeks. The ewe is then started on the OC program shown in Table 8. At the commencement of the program, a 300 mg progesterone CIDR (Eazi-BreedTMCIDR@ sheep insert) is intra-vaginally placed into the ewe. Twelve (12) days later, the ewe begins a shortened treatment for super-ovulation by two initial administrations of follicle stimulating hormone (FSH) (eg administrations at 9.00 am and 5.00 pm of Folltropin@; Vetoquinol Australia). This is then followed up with further FSH administrations on Day 13 as set out in Table 8. On Day 14, the ewe is administered with a 50 pg dose of gonadorelin acetate (GnRH; Fertagyl@ 0.1 mg/ml) and, 6 to 8 hours later, the CIDR is removed. At that time, using surgical laparotomy, follicular fluid is recovered from follicles present in the ovaries using an 18 gauge needle and a 10 ml embryo-safe syringe loaded with 2 ml of a suitable serum-free media for maintaining embryos (eg Vigro Holding Plus; Vetoquinol Australia) treated with heparin (0.1 ml of heparin sodium (1000 iu/ml) per 20 ml vial of media). The recovered fluid is then searched for oocytes, and oocytes present recovered, washed through the serum-free media and incubated at 370 C prior to implantation. This process may or may not involve excising the ovaries from the donor ewe.

[0042] The intermediary recipient ewe ("Egg Box"), synchronised to the oestrus cycle of the donor ewe in a similar manner to that described above in Example 2, is then prepared for implantation by having its ovaries exposed via surgical laparotomy under general anaesthesia and pre-ovulatory follicles are aspirated. These follicles then form the corpus luteum that will maintain the pregnancy following receipt of the donor oocytes (which can also be assisted by administration of GnRH at 18 hours post "CIDR out" and the insertion of a new CIDR (CIDR in) at day 16 to boost progesterone levels). Then, during the surgery, oocytes from the donor are transferred into the recipient ewe's oviduct(s) followed by direct insemination into the uterus.

[0043] Embryo collection from Egg Box ewe and transfer to final recipient ewes On Day 16, a CIDR is intra-vaginally inserted into the Egg Box ewe in preparation for the embryo collection (EC). Then, on Day 20, the Egg Box ewe is flushed (eg using Vigro Complete Flush; Vetoquinol Australia) using standard techniques to collect embryos, and then the embryos are stored or transferred to final recipient ewes (which may be prepared for the ET in accordance with the protocol described above in Example 2, thereby synchronising the oestrus cycle of these animals to the donor ewe (and intermediary recipients)) in accordance with standard ET techniques. Ahead of the embryo transfer, each of the recipient ewes is examined to determine if suitable for receiving embryo(s) by the presence of sound corpus luteum necessary to maintain pregnancy following embryo transfer.

Table 8 - Ewe trial protocol

Day & Time Ewe group Enhanced OC protocol step Day 0 8.00 am Donor CIDR in 8.00 am Recipients CIDR in Day 12 10.00 am Donor 2.5 mls Folltropin 5.00 pm Donor 2.5 mls Folltropin Day 13 9.00 am Donor 2.0 mls Folltropin 9.00 am Recipients CIDR out + 2.0 ml Pregnecol + run with teaser 5.00 pm Donor 2.0 mls Folltropin + Dry out* 5.00 pm Recipients Dry out* two Recipients Day 14 8.00 am Donor 0.5 ml GnRH 10.00 am Donor oocyte collection + remove cidr 11.00 am Dried out Recipients Implant oocytes and insemination to produce "Egg Box" ewes

3.00 pm Egg Box ewes and 0.5 mls GnRH

Final Recipients Day 16 8.00 am Egg Box ewes CIDR in Day 20 8.00 am Egg Box ewes EC surgery 12.00 pm Final Recipients ET

*Dry out = animal taken off food and water prior to surgery

Results and Discussion

[0044] The results of a trial using 10 final recipient ewes yielded 6 healthy lambs. The results are shown in Table 9. The genetics of a special stud ewe no longer able to naturally breed was thereby preserved and expanded.

Table 9 Animals used Donor ewe DG090424 (a special White Suffolk stud ewe no longer able to breed naturally due to major abdominal adhesions) Ram (semen) WR120399 "Egg Box" recipient ewe 09090

Oocytes and embryo numbers Oocytes collected from DG090424: 18 All 18 oocytes were transferred to ewe 09090 (to become the Egg Box) Number of pre-ovulatory Follicles observed (number of transferred oocytes): left 1 (12 oocytes), right 1 (6 oocytes) Embryos collected from 09090: 12 Grade of embryos: 9 x compact morula (cm)- early blastocyst (eb) grade 1 2 x compact morula (cm) grade 2 1 x 3 cell degenerate Embryos transferred to final recipient ewes: 11

Final Ovulations Embryos Received (left-right uterine horn) recipient (left-right) ewes

12072 2-0 2-0 (1 x eb grade 1, 1 x cm grade 2)

12024 0-2 0-1 (1 x eb grade 1)

10488 0-2 0-1 (1 x eb grade 1)

12729 1-0 1-0(1 x eb grade 1)

10682 0-1 0-1 (1 x eb grade 1)

10631 1-1 0-1 (1 x eb grade 1)

10706 0-1 0-1 (1 x eb grade 1)

09602 0-2 0-1 (1 x eb grade 1)

12537 1-0 1-0(1 x eb grade 1)

10698 2-0 1-0 (1 x cm grade 2)

Result: 6 lambs produced

[0045] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0046] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[0047] It will be appreciated by those skilled in the art that the methods of the present disclosure are not restricted in their use to the particular application described. Neither are the methods restricted to their preferred embodiments with regard to the particular elements and/or features described or depicted herein. It will also be appreciated that the methods of the present disclosure are not limited to the embodiment or embodiments disclosed, but are capable of numerous rearrangements, modifications and substitutions without departing from the scope of the methods set forth and defined by the following claims.

Claims (4)

1. A kit comprising at least a container provided with chorionic gonadotropin (Gn) and a container provided with gonadotropin releasing hormone (GnRH) or an agonist thereof, when used in a method of inducing ovulation in a group of sheep for artificial insemination (Al), wherein said method comprises the steps of: providing and/or selecting sheep in oestrus by

(i) treating each sheep within the group with a controlled internal drug (progesterone) release device (CIDR) for a period of 10-14 days; and (ii) removing said CIDR from the sheep and substantially immediately thereafter administering to each sheep, by a single im or iv injection, chorionic gonadotropin (Gn) and, optionally, exposing the sheep to a teaser to induce oestrus; and administering to each sheep in oestrus, at 25-35 hours of removing said CIDR, an amount of gonadotropin releasing hormone (GnRH) or an agonist thereof which is effective to induce synchronised ovulation within a 2 hour window of time.

2. The kit of claim 1, wherein step (i) is conducted by treating each sheep with a CIDR for a period of about 12 days, the chorionic gonadotropin administered in step (ii) is equine chorionic gonadotropin, and the GnRH is administered at about 30 hours of removing said sponge or CIDR.

3. The kit of claim 1 or 2, wherein the GnRH or agonist thereof is administered in an amount effective to induce synchronised ovulation in the oestrus-phase sheep at 48-50 hours of removing said CIDR.

4. The kit of any one of claims I to 3, wherein the step of administering to each oestrus-phase sheep an amount of gonadotropin releasing hormone (GnRH) or an agonist thereof which is effective to induce ovulation comprises administering gonadorelin acetate to each oestrus phase sheep in an amount of 50 g per sheep.