Long term, reversible control of reproduction in dogs

Trigg, T.E., Wright, P.J., Armour, A.F., Williamson, P.E., Junaidi, A., Martin, G.B., Doyle, A.G. & Walsh, J. (2001).
Long term reversible desexing of male dogs and oestrus postponement of bitches, using a GnRH analogue implant. In:
Advances in reproduction in dogs, cats and exotic carnivores (Editors: P.W. Concannon, G.C.W. England, W. Farstad, C.
Linde-Forsberg, J.P. Verstegen & C. Doberska) pp. 255-261 [Journal of Reproduction & Fertility, Supplement 57].
Draft

Use of a GnRH analogue implant to produce reversible,

long-term suppression of reproductive function of male
and female domestic dogs
T.E. Trigg*, P.J. Wright1, A.F.Armour1, P.E. Williamson2, A. Junaidi2†,
G.B.Martin3, A.G. Doyle and J. Walsh
Peptech Animal Health, 35-41 Waterloo Road, North Ryde, 2113, Australia; 1Department of Veterinary
Science, The University of Melbourne, 250 Princes Hwy, Werribee 3030,; 2Division of Veterinary and
Biomedical Sciences, Murdoch University, Murdoch 6150, 3Faculty of Agriculture, University of Western
Australia, Nedlands 6907

* Corresponding author:
35-41 Waterloo Road, North Ryde, NSW 2113, Australia
Phone: +61 2 9870 8788 Fax: +61 2 9870 8787 E-mail: trigg@peptech.com
†
present address - Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia.

Summary

Continuous low-dose administration of a GnRH analogue is capable of postponing oestrus

in bitches and suppressing reproductive function in dogs. A new drug delivery formulation
which could enhance the practicality of this approach for the control of reproduction has
been developed. The objective of this work was to determine if this delivery method could,
by sustained release of the GnRH analogue deslorelin, act as a reversible contraceptive in
domestic dogs of both sexes for periods exceeding one year. Several long-term studies
were performed, monitoring reproductive function in 30 dogs and 52 bitches. Suppression
of reproductive function in males was dose-related, with 14 of 16 dogs receiving deslorelin
doses > 0.25 mg/kg demonstrating suppressed spermatogenesis for more than a year. In
females, the postponement of oestrus for periods of up to 27 months was observed, with no
relationship between the stage of cycle when treatment started and the duration of efficacy.
Treatment-induced effects on fertility were reversible in both sexes. In summary, sustained
release deslorelin implants were shown to elicit reversible long-term reproductive control
in male and female domestic canids.

Introduction

The use of GnRH agonists for reproductive management has been extensively investigated
and has shown great potential (Vickery et al, 1989), but the development of a long-acting
contraceptive has yet to eventuate. Such an agent could be applied to the control of
domestic and wild canid populations and also captive wildlife. Previous development
strategies for a contraceptive for canids that is long-acting, reliable and free of side-effects,
have explored many technologies ranging from vaccination against antigens involved in
the reproductive process (eg zona pellucidae, Mahi-Brown et al 1985; sperm antigens,
Talwar and Naz, 1981; GnRH, Gonzalez et al, 1989) to administration of sex hormones
and their analogues. Each of these approaches has limitations, such as the unreliable

Page 1
 Long term, reversible control of reproduction in dogs

efficacy of many vaccines and side-effects associated with long-term use of steroid
hormones.
GnRH agonists are extremely potent drugs that act by suppressing the function of the
pituitary-gonadal axis, but delivery over a suitable period and in sufficient amounts has
posed a challenge. McRae et al (1985) have reported variously successful attempts to
achieve long-term postponement of oestrus in bitches by delivery of the GnRH agonist
nafarelin using osmotic mini pumps, cholesterol implants, silastic and polylactide co-
glycolide implants.
The major impediment preventing the widespread application of this approach has been the
failure to develop a suitable delivery system with attributes including ease of use, bio-
compatibility, long-term release of sufficient amounts of GnRH agonist and cost
effectiveness. A formulation that meets these criteria, and releases the GnRH agonist,
deslorelin, for extended periods has been developed. This proprietary technology is safe,
efficacious and effective in both sexes, and hence makes long-term shutdown of the
pituitary-gonadal axis in dogs and bitches a practical proposition.
This manuscript describes studies on suppression of reproductive function in male and
female dogs using a novel implant formulation for delivery of deslorelin. Duration of
suppression was examined in relation to dose rate in male dogs. In females the following
were assessed: i) the influence of drug dose, stage of cycle and of pregnancy on postponed
oestrous response; ii) the effect of treatment on pregnant animals; and iii) the interval from
implantation to the next oestrus (postponement of oestrus).

Materials and Methods

Experimental procedures
All treatments and procedures were undertaken under the guidelines of the Animal
Experimentation Ethics Committees of the University of Melbourne and Murdoch
University.
Implants were manufactured by a proprietary method involving extrusion of deslorelin
with a matrix consisting principally of low-melting point lipids and biological surfactant.
The dimensions of a 6 mg implant were 2 cm long and 2.3 mm in diameter; 12 mg was
administered by using two 6 mg implants and 3 mg using half a 6 mg implant. In a real
time dissolution system, these implants released doses of >1µg/day for periods of greater
than one year. Dogs were clipped in the interscapular region and sterile deslorelin-
containing or placebo implants were inserted subcutaneously via single-use syringes.
Routine blood samples were taken, and scrotal circumferences were measured in males.
Similarly for females, plasma samples and vaginal cytology samples were taken according
to the following schedules. At least 3 baseline measurements were established in the 2
weeks prior to implantation, after which, observations and sampling were performed
according to the following timetable: twice weekly for 4 weeks, once per week for 6
weeks, then once every 2 weeks until the return of testes to normal size, or in females until
the onset of pro-oestrus.
Blood samples (10 ml) were collected into heparinised tubes by jugular venipuncture and
immediately placed on ice until centrifugation for 15 minutes at 1000 g. Plasma was
harvested, the sample divided into three aliquots and frozen at –200 C until required for
analysis.
Hormone and GnRH agonist assays
RIA was used for determination of plasma concentrations of testosterone (Hotzel et al,
1995) and progesterone (Gales et al., 1997).
Examination of implantation site.

Page 2
 Long term, reversible control of reproduction in dogs

Tissue was taken from around the implant of eight animals for histological examination,
following restoration of normal steroidogenesis.

Male dogs
Thirty mature dogs of mixed breeding and weight (5-37.5kg) were maintained and housed
in runs, with kennel and shade available at all times. Food was provided once per day in
amounts adequate to maintain body mass, and water was available ad libitum. The animals
were assigned to groups of five dogs on a stratified random basis. Treatment groups: 3, 6,
or 12 mg deslorelin (doses 0.08-0.79 mg/kg), and placebo controls were administered as
subcutaneous implants. Eleven dogs were re-implanted with implants formulated to
contain either 6 or 12 mg deslorelin (dose range 0.26 to 0. 49mg/kg). Three of these eleven
were implanted whilst still suppressed, the other 8 were implanted following recovery of
normal fertility. The restoration of reproductive function was determined from scrotal
circumference and plasma testosterone concentrations (all dogs), semen evaluation (6
dogs), and fertility (2 dogs).

Table 1. The reproductive status and numbers of bitches treated with deslorelin.
Dose of GnRH analogue Total
(mg/dog)
Status 0 3 6 12
Anoestrous 0 1 4 0 5
Dioestrous 10 8 8 9 35
Pregnant 2 0 6 4 12

Female dogs
Fifty-two bitches of mixed breeding and ranging in weight from 5-29.3 kg were treated
with deslorelin, formulated to contain 3, 6 or 12 mg in an sc implant (doses 0.1-2.4 mg/kg),
or were untreated- or placebo-treated controls. They were maintained and housed at three
different kennelling facilities within Australia, in conditions similar to those for the males.
These animals were admitted to the study during pregnancy (at around day 30), mid-
dioestrus, or anoestrus. The animals were examined for signs of pro-oestrus or oestrus
(vulval swelling and discharge, pro-oestrous or oestrous behaviour, changes in vaginal
cells) and jugular blood was sampled for plasma progesterone determination after
implantation (3/w for 2 w, 2/w for 2w then 1/2w until an oestrus was confirmed). The
bitches were euthanased at the end of the study and the reproductive tracts examined.
Vaginal smears were stained with Diff–Quik (Lab-Aids Pty Ltd, Narrabeen 2101 NSW
Australia). The superficial cell index (SCI) was the number of epithelial cells that were
polygonal as estimated from viewing 10 fields (magnification x 40). Nine bitches were
mated at the oestrus which followed oestrus postponement.

Results

No sign of inflammatory reaction was observed at the site of implantation and there was no
associated oedema. A 2-5 cell layer connective tissue encapsulation of the implant was
observed but there were no signs of inflammation past or present.

Dogs
Plasma testosterone
Plasma testosterone was reduced to <1 ng/ml within 6 to 25 days of implantation for all
treatment groups with the exception of one animal (32d, 12mg group)(d = days from

Page 3
 Long term, reversible control of reproduction in dogs

implantation). The mean time (±sem) for testosterone to reach <1 ng/ml was 17.5±1.52
days. The period over which values remained at this level or below ranged from 3 months
to 2.7 years. Duration of effect appeared to be dose dependent, though there was some
individual variation (Fig 1). An example of the effect on plasma testosterone is seen in Fig
2a. Eleven of these dogs were re-implanted with either 6 or 12 mg deslorelin. The effect of
additional treatments on these animals could not be distinguished from the effects of
primary dose of deslorelin.

4.0

3.5

3.0

2.5
Time (years)

2.0

1.5

1.0

0.5

0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Dose deslorelin(mg/kg body weight)

Figure 1. Dose of deslorelin (mg/kg) and the duration of efficacy ( as assessed from
reduction in plasma testosterone) in individual male dogs.
: dogs that were re-implanted once while still suppressed from primary implantation

Restoration of fertility
Restoration of reproductive function was determined from plasma testosterone
concentrations and scrotal circumference measurement (Fig 2b), in all dogs, semen
evaluation in six dogs and fertility in 2 dogs. The semen volume, motility, concentration
and proportion of abnormalities of spermatozoa of the six dogs evaluated were all normal
(Table 2). Two dogs were used to mate bitches in the dog colony and both had at least 1
successful mating which resulted in a pregnancy.

Table 2: Characteristics of semen collected from dogs (n=6) after recovery from
suppression with deslorelin. The range of values for normal dogs is presented for
comparison.
Semen Characteristic Post-recovery values (mean ± sem) Normal range
Volume ejaculate(ml) 1.5 ± 0.4 1 - 25
Spermatozoa
Motility (%) 78.0 ± 3.7 60 - 90
Concentration (x106) 212.8 ± 63.1 20 - 540
Abnormality (%) 2.1 ± 0.3 5 - 20
Alive (%) 78.0 ± 0.2 >50

Page 4
 Long term, reversible control of reproduction in dogs

PW36

PW41
16 PW57

PW62

14 Re-implant PW36 PW75

Scrotal circumference (cm)

PW62, PW75

12

10

6
-10 90 190 290 390 490 590 690 790 890 990 1090 1190 1290
Days (implanted at day 0)

7 PW36
PW41
6
Testosterone Conc. ng/ml

PW57
Re-implant PW36, PW62
5
PW62, PW75 PW75
4

0
-10 90 190 290 390 490 590 690 790 890 990 1090 1190 1290
Days (implanted at day 0)

Figure 2. a) An example of the effect of continuous low level deslorelin on plasma

testosterone in five dogs each given an implant containing12 mg deslorelin. Deslorelin
implant were given at day 0. b) An example of the effect of continuous low level deslorelin
on the scrotal circumference of five dogs each given an implant containing12 mg
deslorelin. Deslorelin implant were given at day 0

Bitches
Stage of oestrous cycle
Table 3 shows the effect of implanting animals at three stages of the oestrous cycle. It was
not possible to distinguish between stage of the oestrous cycle and the duration of efficacy.

Page 5
 Long term, reversible control of reproduction in dogs

All non-pregnant control bitches showed oestrus between 4.5 and 7.5 months from
allocation to their treatment. Pregnant control bitches displayed oestrus between 8 and 10
months post-whelping. Whelping was normal from these pregnant bitches, and viability of
pups was within normal expected range for similar pups born at the research centres.

Table 3. The duration of oestrus postponement (months ± sem) in 52 bitches treated with
GnRH analogue subcutaneously during anoestrus, dioestrus and pregnancy*(nd - no data
collected).
Dose of GnRH analogue (mg/dog)
Status 0 3 6 12
Anoestrous nd * 10 13.9±1.9 nd
Dioestrus 5.5±0.5 10.7±1.4 14.6±3.5 15.5±1.7
Pregnant 8.5±1.5 nd 20.4±3.4 19.6±2.5

Dose
The treatments lengthened the mean inter-oestrous interval at all doses and it was possible
to postpone oestrus for periods of up to 27 months.
Induced-oestrus was observed in all bitches treated during anoestrus, and during dioestrus
when plasma progesterone was <5ng/ml, with the exception of two animals (see Fig 3;
P<0.0001). Signs of pro-oestrus and oestrus were detected 4-8 days after implant
placement in those bitches showing induced oestrus. The treated pregnant bitches did not
show an induced oestrus. At necropsy the presence of intact reproductive tracts was
confirmed.
Of the nine bitches mated at the first oestrus following recovery, six became pregnant. Of
these, 2 were necropsied close to parturition and 4 gave birth to healthy litters. One litter
was euthanased at birth, and 3 bitches raised healthy pups to weaning.
30

25

No Oestrus
20
Progesterone ng/ml

Oestrus

15

10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Figure 3. The effect of plasma progesterone concentration at the time of implantation on

the incidence of induced oestrus in individual bitches following implantation with 3, 6 or
12 mg deslorelin. Each bar represents an individual animal.

Page 6
 Long term, reversible control of reproduction in dogs

Discussion

This study demonstrates that single implant technology using deslorelin as a contraceptive
agent results in safe, long-term, reversible suppression of reproductive function in male
and female dogs. The site of implantation did not demonstrate significant inflammation,
acute or chronic.
Some dogs were suppressed for 600+ days with one implantation and those given a second
implantation whilst still affected by the first implant have shown suppression for >1200
days. In males, the length of effect is related to dose, and with the exception of two
animals, dogs receiving >2.5 mg/kg, were suppressed for a minimum of twelve months.
All dogs returned to normal steroidogenesis following their period of down-regulation.
This was associated with the restoration of testicular volume, a return of sperm
characteristics associated with fertility and an elevation of plasma steroid concentration; a
result in agreement with those of Junaidi et al. (1998). A sample of dogs which were more
closely followed for recovery indicated that, in all cases, normal histological testicular
structure was restored, and the two dogs used to mate bitches following recovery were
successful in achieving pregnancies.
In females, deslorelin administered in continuous low dose postpones oestrus and is safe
when given in mid-pregnancy. As observed previously (Vickery et al, 1989), agonists
often induced oestrus due to an initial stimulatory effect on gonadotrophin release and
gonadal steroid output prior to the suppression of the pituitary-gonadal axis.
No bitch showed an induced oestrus when the minimum plasma progesterone
concentration at the time of implantation circulation was more than 5ng/ml.
This work, together with the findings of Wright et al. (this supplement), who showed
agonist-induced oestrus can be prevented by treatment with progestin, provides a practical
method for contraception of all dogs, irrespective of sex and stage of cycle.

References

Gales NJ, Williamson P, Blackberry MA, and James I (1997) Evidence for a prolonged
postimplantation period in the Australian sea lion (Neophoca cinerea). Journal of
Reproduction and Fertility 111 159-163
Gonzalez A, Allen AF, Post K, Mapletoft RJ and Murphy BD (1989) Immunological
approaches to contraception in dogs. Journal of Reproduction and Fertility Supplement 39
189-198
Hötzel MJ, Walkden-Brown SW, Blackberry MA and Martin GB (1995) The effect of
nutrition on testicular growth in mature Merino rams involves mechanisms that are
independent of changes in GnRH pulse frequency. Journal of Endocrinology 147 75-85
Junaidi A, Williamson P, Cummins JM, Martin GB and Trigg TE (1998) Reproductive
function and pituitary responses to gonadotrophin-releasing hormone (GnRH) in male dogs
treated with the GnRH agonist, deslorelin Proceedings of the Australian Society of
Reproduction and Biology 29 58 (Abstract)
Mahi-Brown CA, Yanagimachi R, Hoffman JC and Huang TT (1985) Fertility control in the
bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and
patterns of estradiol and progesterone levels in oestrus cycles Biology of Reproduction 32 761-
772
McRae GI, Roberts BB, Worden AC, Bajka A and Vickery BH (1985) Long-term reversible
suppression of oestrus in bitches with nafarelin acetate, a potent LHRH agonist Journal of
Reproduction and Fertility 74 389-397
Talwar GP and Naz RK (1981) Immunological control of male fertility Archives of
Andrology 7 177-185

Page 7
 Long term, reversible control of reproduction in dogs

Vickery BH, McRae GI, Goodpasture JC and Sanders LM (1989) Use of potent LHRH
analogues for chronic contraception and pregnancy termination in dogs Journal of
Reproduction and Fertility Supplement 39 175-187
Wright PJ, Verstegen JP, Onclin K, Jochle W, Armour AF, Martin GB and Trigg TE (2000)
Progestin treatment suppresses oestrous responses to the GnRH analogue deslorelin in the
bitch Journal of Reproduction and Fertility Supplement

Page 8