CSIRO PUBLISHING

Wildlife Research, 2008, 35, 548–554 www.publish.csiro.au/journals/wr

The use of a GnRH vaccine to suppress mare ovarian activity

in a large group of mares under field conditions

A. E. Botha A,C, M. L. Schulman A, H. J. Bertschinger A, A. J. Guthrie B, C. H. Annandale A

and S. B. Hughes A
A
Section of Reproduction, Department of Production Animal Studies, University of Pretoria,
Republic of South Africa.
B
Equine Research Centre, University of Pretoria, Republic of South Africa.
C
Corresponding author. Email: ester.botha@up.ac.za

Abstract. The aim of this study was to evaluate the effect of active immunisation against GnRH on ovarian activity and
serum progesterone concentrations in a large group of mares (10 control and 55 experimental) under field conditions as a
model for wildlife species such as zebra and African elephants. Within the experimental group, mares were subdivided into
three age categories: Category 1 (4 years and younger, n = 26), Category 2 (4–10 years old, n = 18), and Category 3 (
11 years
old, n = 11). Experimental mares were vaccinated intramuscularly with 2 mL (400 mg) of the GnRH vaccine Improvac (Pfizer
Animal Health, Sandton, South Africa). Control mares received the same amount of saline solution. The vaccinations were
repeated 35 days later. The ovaries and reproductive tracts of each mare were examined by means of rectal palpation and
ultrasonography on Days 0, 35 and 70. Blood was collected weekly for determination of serum progesterone concentration
until Day 175. On Day 35 after primary vaccination all of the control mares and 14.5% of the experimental mares showed
evidence of ovarian activity on the basis of clinical examination and serum progesterone concentration. On Day 70, all control
mares and none of the experimental mares showed evidence of cyclic activity. No age-related effect within treatment groups
was found. The serum progesterone concentration indicated that all experimental mares remained in anoestrus until Day 175.
Five of the control mares fell pregnant between Days 35 and 70. The five non-pregnant control mares continued to cycle until
the end of the observation period. Having achieved such promising results in this trial we now plan to test the GnRH vaccine in
Burchell’s zebra mares and African elephant cows.

Introduction progesterone or progestagens and the short duration of action

Contraception as a means of population management of captive made their use impractical. A potential negative effect on future
wildlife species is well established. Methods that have been used reproduction after repeated administration of progesterone or
include separation of sexes, surgical techniques and steroid progestagens in prepubertal fillies has also been described
hormone implants. The safety, practicality and reversibility of (Skelton et al. 1991).
methods have enjoyed more attention in recent years, particularly The major alternative to pharmacological manipulation for
where endangered and rare species are involved. Because of long-term control of reproductive cyclicity and behaviour
developments during the last 15 or so years it has become possible involves the surgical removal of both ovaries (ovariectomy).
to successfully apply some methods for the management of free- Ovariectomy is invasive and also associated with various
ranging wildlife populations. Typical examples of these are the complications. After this procedure, some mares may still
use of the porcine zona pellucida (pZP) vaccine to control show signs associated with oestrous behaviour (Ginther 1979;
reproduction in wild horses (Kirkpatrick et al. 1982, 1990; Asa et al. 1980; Hooper et al. 1993). The factors of cost,
Kirkpatrick and Turner 2002) and white-tailed deer (Naugle associated trauma, potential postoperative complications,
et al. 2002; Rutberg et al. 2004). production setbacks and the irreversible loss of breeding
In mares, the control of reproductive activity is a vital potential make this method difficult to implement (D’Occhio
management aspect from both a fertility and behavioural 1993). Ovariectomy is therefore considered undesirable unless
perspective. Anecdotally, oestrous behaviour may negatively circumstances are exceptional.
influence the performance ability of mares engaged in work Recent studies on the use of gonadotropin-releasing hormone
and competition events (Elhay et al. 2007). (GnRH) vaccination for suppression of fertility, aggressive and
In the past, fertility control and the suppression of aggressive sexual behaviour have shown promising results. Vaccination
and sexual behaviour in horses was successfully managed entails the administration of a modified form of the GnRH
by administration of progesterone or progestagens, in oral or hormone in order to stimulate the production of anti-GnRH
injectable formulation, to simulate dioestrus. The high costs and antibodies. These antibodies then bind to the endogenous
the frequent administration associated with the administration of GnRH and inhibit the natural binding of this molecule to its
 CSIRO 2008 10.1071/WR07136 1035-3712/08/060548
Suppressing ovarian activity in mares with GnRH vaccine Wildlife Research 549

receptors on the pituitary gonadotropes. This results in model for contraception of zebra mares and African elephant
suppression of secretion of both follicle-stimulating hormone cows.
and luteinising hormone at the level of the pituitary.
Immunocastration by means of GnRH vaccination as an Materials and methods
alternative to surgical castration to control unwanted male Experimental design
characteristics and behaviour has been studied in male animals
A total of 65 mares, of either barren or maiden reproductive status,
of different species, including bulls (Jago et al. 1997), stallions
of various horse breeds and between 3 and 17 years of age from the
(Malmgren et al. 2001; Stout 2001; Turkstra et al. 2005; Burger
Mounted Unit of the South African Police Services were used for
et al. 2006), Chinese pigs (Zeng et al. 2001), lambs (Janett et al.
this trial. The trial was approved by the Animal Use and Care
2003), rats, dogs and rams (Ferro et al. 2004), African elephants
Committee of the Faculty of Veterinary Science of the University
(Bertschinger et al. 2004; Delsink et al. 2004), cats (Levy et al.
of Pretoria (V068/05). The mares were housed in outdoor
2004; Robbins et al. 2004), puppies (Jung et al. 2005) and feral
paddocks or extensive pastures for the duration of the trial.
swine (Killian et al. 2006).
They were assigned to either a control (n = 10) or an
The use of the commercially available GnRH vaccine
experimental (n = 55) group. For management purposes, all
(Improvac, Pfizer Laboratories, Sandton, South Africa) in
experimental mares were excluded from breeding attempts
boars eliminates boar taint and enhances growth performance
whereas certain control mares were later assigned for breeding.
in uncastrated male pigs (Dunshea et al. 2001; Chumkam and
Within the experimental group, mares were subdivided into one
Ravungsook 2003).
of three age categories: Category 1 (4 years and younger, n = 26),
In stallions and colts the semen quality of animals vaccinated
Category 2 (5–10 years old, n = 18), and Category 3 (
11 years
with a GnRH vaccine deteriorated but the production of semen
old, n = 11).
was never completely suppressed (Dowsett et al. 1996;
Malmgren et al. 2001; Turkstra et al. 2005; Burger et al.
GnRH immunisation
2006). The shedding status of stallions infected with equine
viral arteritis was also converted to non-shedder status in On Day 0, mares from all three age categories of the experimental
vaccinated stallions (Burger et al. 2006). group were injected with 2 mL Improvac (400 mg RnRF-protein
The effect of GnRH vaccination in female animals of various conjugate) (Pfizer Animal Health, Sandton, South Africa)
species (including deer, wild rats and bison) has also been intramuscularly into the left gluteus muscle, whereas all mares
reported. With the use of an early form of a GnRH vaccine it from the control group received 2 mL of a sterile saline solution
appeared that GnRH vaccination is not an effective means of using a 20-gauge, 11/200 needle (Terumo Corporation, Tokyo,
contraception in deer (Becker et al. 1999). However, a larger and Japan). The subsequent booster vaccination (2 mL) was
more comprehensive study on immunocontraception of white- administered into the right gluteus muscle 35 days after the
tailed deer (Miller et al. 2000) showed immunocontraception of primary vaccination (J. F. Kirkpatrick, pers. comm.).
deer to be effective. Female white-tailed deer treated with GnRH
immunocontraceptive vaccine showed fewer oestrus cycles per Transrectal monitoring of the reproductive tract
female than either females treated with a porcine zona pellucida On Day 0, all mares were examined by means of transrectal
(PZP) vaccine or an untreated control group (Curtis et al. 2001). In palpation and ultrasound of the internal genitalia using a 5-MHz
wild rats, GnRH vaccine was found to be more effective than linear array probe (Aloka Echocamera SSD-500, Aloka Mitaka-
mouse zona pellucida peptide vaccine (Miller et al. 1997). shi, Tokyo, Japan) to establish their reproductive status. All
Finally, a single dose of GnRH vaccine prevented pregnancy clinical findings were recorded on a standardised data-capture
in female bison for
1 year (Miller et al. 2004). form.
In mares, the few studies to date report a successful but Ovarian activity was described using mean ovarian volume.
variable effect on suppression of ovarian activity and The length, height and width of the left and right ovaries were
corresponding prevention of oestrous behaviour in adult estimated on transrectal palpation for each mare. These were
mares. These studies have, in most instances, been limited by used to calculate the ovarian volume using the prolate ellipsoid
relatively small numbers of mares: in 10 mares (
2.5 years of age) formula (length  height  width  0.523) (Pavlik et al. 2000).
(Garza et al. 1986), in six mares (2 years of age) (Tshewang et al. The mean ovarian volume for each mare was calculated as the
1997), in four mares (age range: 6–14 years) (Dalin et al. 2002), in average of the left and right ovarian volume. The diameters of
seven mares (Stout et al. 2003), in 18 mares (age range: 3– all ultrasonographically visible follicles
2 cm and the presence
17 years) (Imboden et al. 2006). In only one study was a relatively and number of corporae luteae (CLs) were also recorded.
large population of 48 mares (age range: 3–12 years) studied The dimensions of the uterine horns and body and cervix
(Elhay et al. 2007). In addition, an effect of mare age on treatment were also estimated on transrectal palpation and their palpable
response has been reported in some studies (Dalin et al. 2002; tone was scored. The observed uterine oedema patterns were
Stout et al. 2003), with significant injection-site reactions scored and the presence of intraluminal uterine fluid and other
observed in older mares (Stout et al. 2003). ultrasonographically visible abnormalities were recorded. A
The aim of the present study was to investigate the effects of subjective scoring system was used to grade the degree of
active immunisation against GnRH on reproductive cyclicity in a observed endometrial oedema to reflect changes in circulating
large group of mares of different age categories and to monitor the oestrogen and progesterone concentrations that define oestrus
effect of vaccination and mare age on injection-site reactions. A status (Hayes et al. 1985): 0 (no oedema with a homogeneous
secondary aim of the study was to use the domestic mare study as a echo texture), 1 (the least amount of detectable uterine oedema),
550 Wildlife Research A. E. Botha et al.

2 (moderate amount of oedema throughout), and 3 (most obvious 120 Control

oedema throughout the whole uterus, typical ‘wagon-wheel’ Experimental

Average ovarian volume (cm3)

appearance). The gynaecological data collection was repeated 100
on Days 35 and 70.
80
Blood sampling
Blood samples were collected by means of jugular venipuncture 60
commencing on Day 0 (6 December), approximately the midway
point of the Southern Hemisphere physiological breeding season, 40
and repeated at weekly intervals until Day 175 (29 May), which is
approximately the onset of the non-ovulatory season. The blood 20
samples were collected in plain Vacutainer tubes (BD Vacutainer
Systems, Plymouth, UK) and left to allow clotting. Serum was 0
separated by centrifugation (4000g, 10 min) on the same day as 0 35 70
sample collection. The labelled samples were stored at 20C Days post primary vaccination
until assayed for serum progesterone concentration.
Fig. 1. Mean volume (s.e.) of the ovaries in control and experimental
groups on Day 0 (day of first vaccination), Day 35 (day of second vaccination)
Serum progesterone assay and Day 70.
Assay for serum progesterone concentration were conducted
by means of Radioimmunoassay (Coat-a-Count, Diagnostic Serum progesterone concentration (SPC)
Products Corp, Los Angeles, CA, USA).
The serum progesterone concentrations of the control and
experimental groups over the 175-day observation period are
Injection site reactions
shown in Fig. 2. Within the experimental group there was no
Mares were observed daily in their enclosures by the same effect of age on SPC (P = 0.452) whereas between the two
operator for one week after the vaccinations for injection-site treatment groups there was a significant difference (P < 0.001).
reactions. Reactions at the injection site were scored as follows: 0, Five of the control mares were mated and fell pregnant between
no visible reaction; 1, visible swelling; and 2, visible swelling Days 35 and 70. The remaining five control mares continued to
accompanied by lameness. cycle until the end of the observation period (Day 175).

Statistical analysis Injection-site reactions

All data were analysed using Systat 12 (Systat Software Inc., San The control mares showed no injection-site reactions to the
Jose, CA, USA), using an analysis of variance (ANOVA) of intramuscular saline injections. Table 2 shows the side effects
treatment and age on clinical variables for general linear models to due to GnRH vaccination in experimental mares. The side effects
assess the level of significance. Significance was set at P < 0.05 were transient by nature and by Day 6 were no longer visible. No
age-related effect was observed.
Results
Transrectal monitoring of the reproductive tract Discussion
On Day 0, all control and experimental mares showed clinical The effects of GnRH vaccination on a large group of experimental
evidence of cyclic reproductive activity on clinical examination. mares on both the reproductive tract (Fig. 1, Table 1) and SPC
On Day 35 after the primary vaccination, all control mares and (Fig. 2) were observed. These effects were shown to be rapid in
only 8 of 55 (14.5%) experimental mares showed evidence of onset and statistically significant.
ovarian activity on clinical examination. On Day 70 after the Thirty-five days after the primary vaccination, only eight
primary vaccination, all control mares and none (0%) of the 55 (14.5%) of the experimental mares showed evidence of cyclic
experimental mares showed evidence of ovarian activity on ovarian activity. By Day 70 (35 days after the booster vaccination)
clinical examination. There was no difference between the all experimental mares showed suppressed ovarian activity. For
volumes of the left and right ovaries within individual mares this trial cyclic inactivity was characterised by the absence of any
and therefore the mean ovarian volume of each animal at each follicle >2 cm in diameter, no visible CL and SPC <1 nmol/L. This
examination was subjected to further statistical analysis. The effect was significant (and bilateral, as described by Imboden
mean ovarian volumes of both groups of mares on the days of et al. 2006), with a marked decrease in the mean ovarian volumes
primary (Day 0) and booster (Days 35) vaccinations and on Day as well as in concurrently observed changes to the dependant
70 of the trial are shown in Fig. 1. variables characterising ovarian structures and the tubular
There was a significant treatment effect on ovarian volume genitalia (cervix and uterus). The ovaries were uniformly small
between the control and experimental mares; no age effect was and apparently inactive with follicular atresia, similar to
seen within the experimental groups. All clinical variables of the seasonally anoestrus mares (Dalin et al. 2002; Elhay et al.
reproductive tract observations recorded are summarised in 2007). The tubular genitalia also showed atrophy typical of
Table 1. seasonal anoestrus. Two GnRH vaccinations (Improvac)
Suppressing ovarian activity in mares with GnRH vaccine Wildlife Research 551

Table 1. The number of control and vaccinated mares allocated by clinical findings at the time of examinations
C, control mares; A1, Age Category 1 (4 years); A2, Age Category 2 (5–10 years); A3, Age Category 3 (
11 years)

Clinical variable Day 0 Day 35 Day 70

category C A1 A2 A3 C A1 A2 A3 C A1 A2 A3
(n = 10) (n = 26) (n = 18) (n = 11) (n = 10) (n = 26) (n = 18) (n = 11) (n = 10) (n = 26) (n = 18) (n = 11)
Uterine tone
0 4 9 9 4 3 21 13 8 1 26 16 8
1 6 11 9 5 4 4 2 2 4 0 1 3
2 0 6 0 2 3 1 3 1 4 0 1 0
3 0 0 0 0 0 0 0 0 1 0 0 0
Cervix tone
0 2 8 2 2 1 21 12 8 1 26 16 8
1 8 12 8 7 4 4 5 2 4 0 2 3
2 0 6 8 2 4 1 1 1 4 0 0 0
3 0 0 0 0 1 0 0 0 1 0 0 0
Uterine oedemaA,B
0 4 12 11 5 6 26 17 10 1 26 18 11
1 3 8 5 4 3 0 1 1 2 0 0 0
2 2 2 2 2 1 0 1 0 1 0 0 0
3 1 2 0 0 0 0 0 0 1 0 0 0
Follicle >2 cm
Yes 9 20 8 5 9 2 0 1 8 0 0 0
No 1 6 10 6 1 24 18 10 2 26 18 11
CL present
Yes 8 21 13 11 5 2 1 2 9 0 0 0
No 2 5 5 0 5 24 17 9 1 26 18 11
A
Two datasets were missing for uterine oedema with examination at Day 0, Age Category A1.
B
Five control mares were diagnosed pregnant and uterine oedema score for Day 70 of the five control mares not scored.

100

Control
Experimental

80
Progesterone concentration (nmol/l)

60

40

20

0
0 35 70 105 140 175
Days post primary vaccination

Fig. 2. Progesterone concentrations (s.e.) of control and experimental mares. Arrows indicate times of
vaccinations. The encircled data points reflect raised mean SPC obtained from the control mares because five
mares were in the early stages of pregnancy.
552 Wildlife Research A. E. Botha et al.

Table 2. Injection-site reaction scores (number and percentage) after GnRH vaccination in experimental mares (n = 55)

Score = 0 Score = 1 Score = 2

Age category 1 2 3 1 2 3 1 2 3
Primary vaccination (n) 26 18 11 0 0 0 0 0 0
(%) (100) (100) (100) (0) (0) (0) (0) (0) (0)
Booster vaccination (n) 20 17 10 4 1 1 2 0 0
(%) (76.9) (94.4) (90.9) (15.4) (5.6) (9.1) (7.7) (0) (0)

35 days apart apparently resulted in the effective downregulation The details of the Improvac vaccine are unknown to the
of ovarian activity in all 55 experimental mares. authors as they constitute proprietary information with a single
The small percentage of mares that still showed evidence of exception that the antigen is a RnRF-protein conjugate. Equity
ovarian activity and progesterone secretion after the initial (Pfizer Animal Health, West Ryde, NSW, Australia) is
vaccination is probably due to individual variation in cyclic manufactured by the same company and consists of a GnRH
status at the time of initial vaccination. The eight mares peptide conjugated to a protein carrier combined with an
(14.5%) that were still cyclic 35 days after the initial immunostimulating complex consisting of Saponin Quil A,
vaccination were all in the luteal phase at the time of the cholesterol and dipalmitoylphosphatidyl choline as adjuvant
vaccination (on the basis of both clinical findings and SPC (Elhay et al. 2007). The results of our trial to date, however,
values of >1 nmol/L). This supports the findings that the stage indicate better and more consistent results than those reported
of oestrous cycle and nature of ovarian structures at first with Equity.
administration of vaccine is of importance in terms of the No adverse effects were observed in the experimental mares
observed response (Tshewang et al. 1997; Dalin et al. 2002; after the initial vaccination, which is consistent with the findings
Imboden et al. 2006). described by Dalin et al. (2002), but contrary to the findings of
The SPC was shown to provide a reliable indication of ovarian Imboden et al. (2006), where injection-site reactions occurred
activity for both group and individual monitoring of the response after the initial vaccination. The few animals displaying injection-
to GnRH vaccination of a relatively large number of mares. The site reactions during this study were observed only after the
SPC clearly characterises the effect of GnRH vaccination on the booster vaccination. All adverse reactions were found to be
ovarian activity of the mares. Following the primary vaccination, transient and mild, and by Day 6 were no longer visible. This
eight mares still showed signs of ovarian activity, whereas after is in contrast to the findings of Imboden et al. (2006), where the
the second vaccination no luteal activity was evident. Baseline adverse effects to the vaccination with Improvac were found to be
SPC persisted until the end of the observation period on Day 175. quite severe. This can possibly be due to the fact that in the present
Once again, there was no age effect within the experimental study all injections were administered intramuscularly into the
group, contrary to the findings of Tshewang et al. (1997) and gluteal muscles (J. F. Kirkpatrick, pers. comm.) and not into the
Dalin et al. (2002). On Days 42, 49 and 56 there were 3, 1 and 1 neck, as described by Imboden et al. (2006).
mares, respectively, with SPC >1 nmol/L. Thereafter, and until This study showed a close relationship between the observed
the end of the observation period, SPC of all experimental mares clinical response and SPC within a large group of mares. This
remained <1 nmol/L. This is well below the threshold value of finding supports the conclusion that SPC provides an effective
6 nmol/L quoted by Elhay et al. (2007). In comparison, all five method for monitoring the response to GnRH vaccination in horse
non-pregnant control mares showed SPC indicative of ovarian mares of all ages.
cyclicity. On the basis of the results of our trail in mares, Improvac is a
Five of the control mares became pregnant during the course of promising prospect for the contraception of zebra, African
the observation period. This would explain the large increase in elephants and perhaps other wildlife species. The vaccine is
standard error obtained between Days 80 and 110. While the non- freely available, cheap, stored at 4C and no further
pregnant control mares continued to cycle until the end of the preparation (e.g. mixing of antigen and adjuvant) is required
observation period (Day 175), none of the experimental mares before injection. Side effects at the chosen site of injection were
resumed cyclicity. This probably reflects antibody titres that were minimal. Although of a slightly viscous nature, the vaccine has
still sufficiently high to neutralise endogenous GnRH. As a result, been successfully administered remotely via darting in African
insufficient gonadotropins were available to resume normal elephant bulls with both Dan Inject (Børkop, Denmark) and Pneu-
ovarian activity. The relatively late stage of the physiological Dart (Williamsport, PA, USA) darts (H. Bertschinger, pers.
equine breeding season may also have contributed to this comm.). Reliable reversibility of anoestrus induced by
observation. Improvac has yet to be investigated. Imboden et al. (2006)
The efficacy achieved with the Improvac GnRH vaccine in our were unable to establish this because the mares were no longer
trial was greater than that stated in earlier reports (Garza et al. accessible after the initial phase of the trial. Elhay et al. (2007)
1986; Tshewang et al. 1997; Stout et al. 2003). Likely established a 100% return to folliculogenesis 8–28 weeks after the
explanations for this are improvements in the antigen and second vaccine with Equity. The mares in the present study will
adjuvant used in the final vaccine formulation that evoke a continue to be monitored during the 2007–08 breeding season and
better, more consistent and longer-lasting immune response. the data regarding reversibility of Improvac contraception will
Suppressing ovarian activity in mares with GnRH vaccine Wildlife Research 553

become available in the near future. A likely advantage of a GnRH Dalin, A. M., Andresen, Ø., and Malmgren, L. (2002). Immunization against
vaccine used for contraception of wildlife species such as African GnRH in mature mares: antibody titres, ovarian function, hormonal levels
elephant and zebra is induced anoestrus. The females of species and oestrus behaviour. Journal of Veterinary Medicine 49, 125–131.
doi: 10.1046/j.1439-0442.2002.00427.x
like African elephants and zebra, once they have reached
Delsink, A., Bertschinger, H. J., Kirkpatrick, J. F., DeNys, H., Grobler, D., van
reproduction age, spend most of their lives in a state of
Altena, J. J., and Turkstra, J. (2004). Contraception of elephant cows
anoestrus. African elephants have an average intercalving in two private conservancies using porcine zona pellucida vaccine, and
interval of four years. Of this, 22 months is accounted for by control of aggressive behaviour in elephant bulls with a GnRH vaccine. In
pregnancy and approximately two years by lactation anoestrus ‘Proceedings of an Expert Consultation on the Control of Wild Elephant
(Brown et al. 2004). This means that cows usually conceive Populations’. pp. 69–72. (Utrecht University: The Netherlands.)
during the first oestrus once cycling resumes. Thus, an elephant Delsink, A. K., van Altena, J. J., Grobler, D., Bertschinger, H., Kirkpatrick,
cow probably comes into oestrus approximately only once every J. F., and Slotow, R. (2006). Regulation of a small, discrete African
four years. The use of pZP immunocontraception in feral horses elephant population through immunocontraception in the Makalali
(Kirkpatrick et al. 1990) and African elephants (Delsink et al. Conservancy, Limpopo, South Africa. South African Journal of
Science 102, 403–405.
2006) causes infertility by interfering with fertilisation and the
Dowsett, K. F., Knott, L. M., Tshewang, U., Jackson, A. E., Bodero, D. A., and
immunised females continue to exhibit oestrous cycles. The Trigg, T. E. (1996). Suppression of testicular function using two dose rates
authors of the present study hypothesise that pZP is more of reversible water soluble gonadotropin-releasing hormone (GnRH)
likely to result in behavioural and social disturbance because vaccine in colts. Australian Veterinary Journal 74, 228–235.
individuals in herds are continually attracting males as a result of Dunshea, F. R., Colantoni, C., Howard, K., Mc Cauley, I., Jackson, P. et al.
coming into heat. Comparative studies are needed to test this (2001). Vaccination of boars with GnRH vaccine (Improvac) eliminates
hypothesis. boar taint and increases growth performance. Journal of Animal Science
79, 2525–2535.
Elhay, M., Newbold, A., Britton, A., Turley, P., Dowsett, K., and Walker, J.
Acknowledgements
(2007). Suppression of behavioural and physiological oestrus in the mare
We express our gratitude to the veterinary and other personnel of the South by vaccination against GnRH. Australian Veterinary Journal 85, 39–45.
African Police Services and the Section of Reproduction, University of doi: 10.1111/j.1751-0813.2006.00092.x
Pretoria, for their valued contributions during the course of this trial. Ferro, V. A., Khan, M. A. H., McAdam, D., Colston, A., Aughey, E., Mullen,
A. B., Waterston, M. M., and Harvey, M. J. A. (2004). Efficacy of an anti-
fertility vaccine based on mammalian gonadotrophic releasing hormone
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j.jri.2004.08.004 Manuscript received 6 September 2007, accepted 20 February 2008

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