Studies on the use of Ovuplant
Induction of ovulation at a precise time may be an advantage to the equine practitioner for the following reasons: (a) to ensure ovulation occurs within 36-48 h of mating to stallions with normal fertility and thus prevent rebreeding, (b) appointment servicing for mares transported to stallions, (c) to ensure a single insemination for a problem broodmare, (d) to ensure ovulation close to the time of artificial insemination of cooled transported or frozen semen or for mares mated to stallions with low fertility associated with poor sperm longevity, and (e) to separate inseminations when many mares are presented in oestrus at the same time.
Initially human chorionic gonadotrophin (hCG) was the only commercially available preparation for induction of ovulation at 36-48 h when administered to a mare in early oestrus with a 30-40 mm follicle. Drawbacks to the use of hCG are that it is of human derivation which may influence purity or supply and refractoriness has been noted after multiple treatments associated with antibody formation.
An alternative agent to hCG for the induction of ovulation such as GnRH or analogues may be beneficial for the following reasons: (a) its smaller molecular weight make it less antigenic thus allowing multiple administration within the same breeding season while still reliably inducing ovulation, (b) it may be used in alternate cycles with hCG, (c) because it is a pure synthetic product there is no probability of viral contamination, and (d) supply would not be related to availability of pregnant women.
In this study below we examined whether an analogue of GnRH (deslorelin-“Ovuplant”) released from an implant in a controlled fashion over at least 72 h could consistently induce a luteinising hormone (LH) surge capable of predictably hastening ovulation.
1) A. O. McKinnon, A. M. Nobelius, S. T. D. Figueroa, J. Skidmore, J. R. Vasey, and T. E. Trigg. Predictable Ovulation in Mares Treated with an Implant of the GnRH Analog Deslorelin. Equine.Vet.J. 25:321-323, 1993.
The experimental animals were part of a herd of 95 mares in good body condition, kept on pasture and given lucerne hay and grain supplement as necessary. They were studied between October 1990 and March 1991 at the Goulburn Valley Equine Hospital.
Mares were teased daily and ultrasonography was used to monitor follicular size and to determine ovulation and number of ovulations per cycle. Mares were examined every 3 days during dioestrus, daily during early oestrus and twice daily (06.30 and 18.30 h) when a follicle > 30 mm in diameter was detected. Ovulation was diagnosed by absence of a previously identified pre-ovulatory follicle (>30 mm diameter) and visualisation of the characteristic echogenic structure formed by the collapse of the follicular walls. All cycles were induced by luteolysis effected by administration of PGF2 on Days 7-10 after ovulation from mares that had follicles < 20 mm in diameter.
Only 61% of mares showed oestrus at some time before assignment. But 87% of mares became oestrus at some time before ovulation. This percentage was not markedly different from that expected and can be used to illustrate difficulties involved with assigning mares on teasing data only. In the absence of behavioural oestrus, assignment was accurately performed on follicle size, relaxing cervix and presence of endometrial folds.
Mean time to ovulation in higher doses of GnRH analogue (Ovuplant) were not different from that in hCG and both drugs significantly decreased time to ovulation compared to controls. These results suggested that the appropriate dose and release profile of the GnRH analogue (deslorelin) was equally efficacious in inducing ovulation in cyclic mares when compared with hCG and were useful in deciding if other international studies would be commissioned. Other studies confirmed these findings and later the drug was released for commercial use.
Since the release of Ovuplant it has been widely accepted by veterinarians as a reliable ovulating induction agent. It has saved many mares from being re-bred when for a variety of reasons it was not possible to accurately predict the precise time of ovulation.
After the initial release and acceptance of the breeding industry of the new drug, it became apparent that other possible uses should be explored.
Mares in winter anoestrous have reduced gonadotropin releasing hormone (GnRH) synthesis and storage in the hypothalamus and decreased quantities of luteinising hormone (LH) in the anterior pituitary. Induction of ovulation in seasonally anovulatory mares in deep winter anoestrous cannot be recommended on commercial breeding farms due to 1) poor response to GnRH or analogues or multiple injections of equine pituitary extracts, or 2) failure of the mare to maintain the corpus luteum (CL) that resulted from induced ovulations. In one study it was concluded that constant administration of GnRH agonists may induce ovulation in mares during seasonal anoestrus; however, percentage of mares ovulating and the lack of reproducibility of effect indicated that this approach was inappropriate for use as a reliable method to manipulate breeding activity in commercial broodmares. Pulsatile administration of GnRH produced better results compared to constant infusion or absorption.
More success is obtained however, when mares enter the transition period when the combined effects of photoperiod, nutrition and warmth, result in increased GnRH synthesis and storage. Progression through the transition period can be monitored hormonally or by determining the follicular population with ultrasonography. Expected response of early or mid transition mares to constant infusion of GnRH or analogues is 50-75% ovulation rate. Most mares will maintain the CL and late in the transition, mares respond more predictably. The GnRH analogue Deslorelin (“Ovuplant”) has been shown effective in increasing LH and predictably hastening ovulation in cycling mares. Published LH profiles in cycling mares suggest a dose dependent response with maximal effect in 12-24 hours and duration of 3-4 days after implantation with Ovuplant.
The report below describes our experiences with experimental administration of Ovuplant to mares in the transition period prior to their first ovulation of the breeding season.
2) A. O. McKinnon, J. R. Vasey, T. B. Lescun, and T. E. Trigg. Repeated use of a GnRH analogue deslorelin (Ovuplant) for hastening ovulation in the transitional mare. Equine.Vet.J. 29 (2):153-155, 1997.
The experimental animals were part of a herd of 55-65 mares in fair to good body condition, kept on pasture and given lucerne hay and grain supplement as necessary. The studies were performed between July and November in 1994 (experiment 1) and 1995 (experiment 2) at the Goulburn Valley Equine Hospital, Victoria, Australia.
Mares were teased daily and ultrasonography was used to monitor follicular size, determine ovulation and monitor the CL. Mares were examined every 2 days during transition and then daily after assignment to treatment. Ovulation was diagnosed by absence of a previously identified pre-ovulatory follicle (>30 mm diameter) and visualisation of the characteristic echogenic structure formed by the collapse of the follicular walls. Whenever possible mares were monitored to determine the time of the subsequent ovulation (inter-ovulatory interval).
The experiments were conducted over two breeding seasons. Mares were not bred.
In 1994, 21 early transitional mares were randomly assigned to either a control (n=10) or treated (n=11) group on the same day (September 29). Treated mares were implanted every other day with one implant of Deslorelin until either ovulation had occurred or six implants had been administered.
In 1995, 20 late transitional mares were randomly assigned to either control or treatment groups after they were identified with a ³ 30 mm follicle, were in heat and had endometrial folds demonstrated using ultrasonography. Treated mares were implanted every other day as above. Implants were placed subcutaneously in the neck in both experiments.
In 1994, utilising mares in early transition, we were able to demonstrate an apparent effect of the implants in hastening ovulation (ovulation in <10 days) in treated mares (6/11) versus controls (0/10). However, the mean ovulation date was not different between groups and reflected the effect of 5 mares that did not ovulate after treatment. Those 5 mares may have underwent some form of “down regulation” in GnRH receptors as their follicular size decreased quite clearly in comparison to control mares. This experiment highlighted the difficulty in selecting mares randomly for treatment during the early part of the transition period. The responses are similar to previous reports with GnRH therapies early in the transition.
In experiment 2 in 1995, we were able to demonstrate the ability of the implants to hasten ovulation late in the transition. The number of mares with a ³ 30 mm follicle ovulating within 3 days of treatment (8/10) suggests that the procedure would be useful in predicting ovulation in transitional mares. Failure of mares to ovulate during transition into the breeding season is a major source of frustration to clients, breeding farm managers and veterinarians. The average number of implants needed was 2.1 and this may result in savings in decreased veterinary costs, decreased boarding charges, decreased teasing and handling and better usage of the stallion.
Each year, one mare in the treated group became anoestrus or transitional again and thus demonstrated ovulation may have been forced upon them prematurely. From this perspective it would appear prudent to wait until a transitional mare has demonstrated maturity of the reproductive axis by maintaining large (>30mm) follicles for a few days before initiating induction of ovulation. The increased inter-ovulatory interval detected in treated mares in experiment 2 may have been associated with premature entry into the cyclic season or may suggest a leuteotropic action of the implants. No progesterone measurements were taken and would be a logical area for further investigation.
In summary, it was demonstrated that accurate, timed ovulation was achieved during the late transition using multiple implants of Ovuplant.. This regime is simple, may be cost effective and is expected to be useful for practicing veterinarians.
At this stage no studies had actually looked at what the expected time to ovulation (in hours) was after administration. This has important ramifications (see below). We decided that we should take a close look at some of the effects that various drugs may have on the reproductive cycle, in particular on time to ovulation.
3) A.O. McKinnon, W.J. Perriam, T.B. Lescun, J. Walker, J.R. Vasey and T.E. Trigg*. Effect of a GnRH analogue (Ovuplant), hCG and dexamethasone on time to ovulation in cycling mares. World Equine Veterinary Review Vol 2 page 16-18, 1997.
Induction of ovulation may be an advantage to the equine practitioner for the reasons listed above: In addition, induction of ovulation at a precise time is necessary for oocyte retrieval in gamete intrafollopian transfer (GIFT) in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) programmes.
Most studies have examined hastening ovulation, however there appears to be little information on delaying ovulation. Delaying ovulation would be particularly useful when organising transport of cooled semen. One study found that by administering the synthetic glucocorticoid, dexamethasone (30 mg/day) in midcycle (day 10 from ovulation) that only 1 of 8 mares exhibited behavioural oestrus, compared to 7 of 8 control mares and ovulation occurred in 1 and possibly in 2 treated mares, compared to all 8 control mares. In addition there was a significant reduction in mean maximum luteinizing hormone (LH) concentration and follicle size.
Studies on hastening ovulation using human chorionic gonadotrophin (hCG) began in the 1940’s. Human chorionic gonadotrophin reliably induces ovulation at 36-48 h when administered to a mare in early oestrus with a 30-40 mm follicle, however some question the use of repeated hCG in consecutive cycles as refractoriness has been noted after multiple treatments, perhaps associated with antibody formation. A GnRH analogue, “Ovuplant”, has been effective as an alternative for induction of ovulation in cycling mares and transitional mares. Apparently only two studies have directly compared hCG and Ovuplant and reported an ovulation interval of 1.88 and 1.98 days and 43 and 46.9 h respectively. However both those and other studies have allowed an interval of 12 hours between examinations.
The purpose of this study was to examine the effect of dexamethasone on delaying ovulation when administered to mares with a mature follicle and in oestrous and to more accurately characterise the time interval from administration to ovulation with Ovuplant and hCG.
When mares were detected with a follicle of ³ 30 mm, a softening cervix and endometrial folds, they were assigned to one of three treatments 1) Treatment intramuscularly every 12 hours until ovulation with 20mg dexamethasone (n=10 cycles), 2) Single subcutaneous administration of Ovuplant (n=10 cycles) and 3) No treatment (n=10 cycles). Mares were examined for ovulation every twelve hours.
Using the same assignment criteria as Experiment 1, mares were treated with either 2500 IU hCG intravenously (n=26 cycles) or a single subcutaneous implant of Ovuplant (n=26 cycles). Mares were examined with ultrasonography for ovulation at least every 2 hours from ~ 28 h after administration.
Dexamethasone did not increase time to ovulation compared to controls. Ovuplant administration resulted in a significant decrease in time to ovulation (40.5 ± 6.2 h) compared to either controls (61.3 ± 31.4 h) (P<0.05) or dexamethasone treatment (54.6 ± 16 h) (P<0.05).
Time to ovulation was significantly different (P<0.001) between hCG (35.9 h ± 3.8 h) compared to Ovuplant (40.7 ± 3.2 h) (Figure 1).
Data was removed for ovulation < 24 h from three cycles in hCG treated mares and 2 cycles in Ovuplant treated mares.
Techniques to delay time to ovulation would be of benefit to practitioners and researchers by allowing delayed breeding or treatments. Dexamethasone was not able to delay ovulation as used in this study. Other techniques that may be considered are antisera against either the pituitary, follicle stimulating hormone (FSH), oestrogen, luteinizing hormone or even GnRH, or administration of inhibin, follicular fluid or progesterone. For any substance to be useful and practical it would need to be effective late in the cycle, after selection of the preovulatory follicle had occurred.
The finding of significant difference in time to ovulation of Ovuplant versus hCG has important clinical ramifications for those needing accurate ovulation times. At the GVEH we have used Ovuplant for breeding mares to frozen semen in the last three breeding seasons. A total of 26 and 54 mares were bred for a total of 39 and 98 cycles in the 1995 and 1996 breeding seasons. Whenever possible if hCG had been used the previous cycle, Ovuplant was used the next, however due to the recognised difference in time to ovulation between the two drugs, most commonly Ovuplant was administered. In our hands Ovuplant was administered at 6-7 P.M. on one day and then the mare next examined at + 24 h, again at +36 h (6-7 A.M.) and then hourly as indicated until ovulation. With this regime the mares ovulated during hours when there were many people available to handle and examine them. When using hCG to mimic the same examination schedule (daylight hours) the drug had to be administered at 12 midnight and then the mares examined at +12 h, +24 h, +31 h (7A.M.) and then hourly as indicated.
Later in the breeding season it became even more critical to know the expected time of ovulation for in vivo matured oocyte collection for ICSI experiments. In this study, due to time constraints, mares were all subjected to follicular aspiration at 39 hours after Ovuplant. A total of 59 mare cycles were programmed for follicular aspiration. On 10 occasions (17%) ovulation had occurred prior to the time of aspiration, sometimes between identification of the preovulatory follicle and the aspiration attempt. Thirty two oocytes were recovered from 49 attempts (65%). Oocyte recovery was different ( P< 0.001 ) for matured irregular follicles (30/38-79%) versus not matured follicles (2/11-18%).
Knowing the time of expected ovulation will have profound influences on accuracy of programming mares to ovulate at times were personnel of maximum experience and efficiency are available. Detected differences in time to ovulation of these two drugs will enable clinicians to determine which compound is most appropriate to be administered given the availability of personnel and time management.
Number of mares responding (ovulating) to either hCG or Ovuplant as a function of time (values grouped into 2 hourly increments and the curve smoothed).
Portions of the studies reported above were funded by RIRDC (Australia).
Ovuplant has been demonstrated to be effective to accurately predict the time of ovulation and has resulted in an increased awareness amongst the horse breeding fraternity that one breed per cycle is all that is necessary. We (GVEH) aim at a number of serves per cycle of 1.05. In other words we expect that after 100 mare cycles the stallions will have to serve 105 times. This has critical ramifications for stallions with poor libido, low sperm numbers and shuttle stallions with limited services per season. Using Ovuplant as outlined above will help achieve these objectives.
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