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608
A.M.
JOURNAL
GLAZIER
OF EXPERIMENTAL ZOOLOGY 283:608–611 (1999)
RAPID COMMUNICATIONS
Time of Ovulation in the Brushtail Possum
(Trichosurus vulpecula) Following PMSG/LH
Induced Ovulation
A.M. GLAZIER*
Manaaki Whenua–Landcare Research & Cooperative Research Centre for
Conservation and Management of Marsupials, Lincoln, Canterbury,
New Zealand
ABSTRACT
This study aimed to determine the timing of ovulation in response to a new induced ovulation regime for the monovulatory brushtail possum (Trichosurus vulpecula). Ovarian
stimulation was achieved by a single subcutaneous injection of 15 IU pregnant mare serum gonadotrophin (PMSG). This treatment resulted in promotion of a large number (9–16) of Graafian
follicles >2mm diameter on the ovaries. Seventy-two hours later a single intramuscular injection
of 4 mg porcine luteinizing hormone (LH) was administered to induce ovulation. Groups of possums were killed 24 hr post-LH injection and subsequent groups were killed at 6-hr periods up to
48 hr later. Ovulation occurred from 30 hr to 42 hr post-LH. The ovulatory success increased from
3% at 30 hr post-LH to 83% at 48 hr post-LH. Oocytes were recovered primarily from the oviducts
at 36 hr post-LH. Thereafter oocytes were recovered increasingly from the uteri and by 48 hr postLH, were only found there. The implications of these observations for artificial breeding in possums are discussed. J. Exp. Zool. 283:608–611, 1999. © 1999 Wiley-Liss, Inc.
Induced ovulation of Australian marsupials has
recently attracted increased attention due to interest in applying new reproductive technologies
to conservation of endangered species and in the
management of problem populations (Rodger, ’97).
In New Zealand, the introduced Australian brushtail possum (Trichosurus vulpecula) is considered
a major pest species (Livingstone, ’91). Manipulation of its breeding, through biological control
of fertility (immunocontraception), is regarded as
a sustainable and humane method of controlling
its population (Jolly, ’93). Such a method has facilitated the development of a suitable induced
ovulation protocol (Glazier and Molinia, ’98) to
provide multiple mature oocytes for screening of
candidate contraceptive antigens (Rodger, ’97). Alternatively, it would also improve our ability to
manipulate marsupial reproduction in captivity
for the study of early development and allow development of in vitro fertilization (IVF) techniques, using the possum as a model species
(Mate, ’98).
The need to accurately determine the time of
ovulation in response to induced ovulation protocols developed for the monovular possum (Rodger
and Mate, ’88; Glazier and Molinia, ’98b), is paramount for the development of artificial manipulation of possum reproduction (Rodger, ’90; Molinia
© 1999 WILEY-LISS, INC.
et al., ’98a). Although some of the aspects of normal ovulation in the possum are understood
(Tyndale-Biscoe and Renfree, ’87), the precise timing of ovulation is unknown.
Rodger and Mate (’88) suggested that ovulation
in possums stimulated with PMSG/GnRH started
at about 24 hr post-GnRH, a similar time to that
in the tammar wallaby (Macropus eugenii) which
has an LH surge reaching a peak 25 min after
GnRH injections (Evans et al., ’80), with ovulation occurring as early as 20 hr later (Sutherland
et al., ’80; Harder et al., ’85). A recent study in
the tammar (Molinia et al., ’98b) suggests that
ovulation occurs as late as 36 hr post-LH when a
FSH/LH induced ovulation protocol is used.
Recently, the induced ovulation method for possums of Rodger and Mate (’88) was improved by
increasing the dose of PMSG and replacing GnRH
as the ovulatory stimulus with porcine LH (Glazier and Molinia, ’98). This resulted in the promotion of increased numbers of Graafian follicles
>2 mm diameter and subsequently improved re-
*Correspondence to: A.M. Glazier, Manaaki-Whenua, Landcare Research NZ Ltd, P.O. Box 69, Lincoln, Canterbury, New Zealand. Email: glaziera@landcare.cri.nz
Received 1 June 1998; Accepted 3 November 1998.
TIME OF OVULATION IN THE POSSUM
609
covery of oocytes. The present study was designed
to investigate the time of ovulation using this
optimised PMSG/LH induced ovulation protocol
(Glazier and Molinia, ’98) so that the time of artificial insemination (Molinia et al., ’98a) or of recovery of fertilizable oocytes could be better
predicted.
of these follicles were measured by the eyepiece
graticule as above. The ovulatory success (OS) was
calculated as N/(N1 + N) × 100 where N = number of ovulation’s per animal and N1 = number of
remaining large (>2 mm diameter) follicles per
animal, expressed as a percentage.
MATERIALS AND METHODS
Values are presented as means and mean % ±
standard errors of the mean. Data from the study
on the time of ovulation in response to LH were
analysed by a one-way ANOVA with a post-hoc
test on pairs of means using Tukey’s HSD procedure (Wilkinson, ’89), and data on the site of oocyte recovery and mucoid thickness are expressed
as percentages.
Animals
Possums were trapped locally and transported
to the Landcare Research Animal Facility at Lincoln. On arrival they were weighed, their general
condition was assessed, and they were housed in
individual cages with access to water ad libitum
and were fed a diet of fresh fruit, vegetables and
cereal pellets. All animals used weighed between
2 and 3 kg and had a well-developed pouch indicating sexual maturity.
All experiments were performed in accordance
with the 1987 Animals Protection (Codes of Ethical Conduct) Regulations of New Zealand, and approved by the Animal Ethics Committee, Landcare
Research.
Induced ovulation
Female possums (n = 6 per treatment group)
were given a single intramuscular injection of 15
IU PMSG (Folligon: Intervet, Oss, The Netherlands)
dissolved in sterile water. Seventy-two hours later
they were treated with a single intramuscular injection of 4 mg porcine LH (Lutropin-V, Vetrepharm,
Australia). Animals were anaesthetised by administration of O2/CO2 mixture (1:3) and then killed at
24, 30, 36, 42, and 48 hr post-LH injection by a
single intracardiac injection of 1.5 ml sodium
pentabarbitol (300 mg/ml). Reproductive tracts were
then removed whole.
Statistical analysis
RESULTS
Ovulation rate with time
At 24 hr post-LH, no ova were recovered and
ovaries contained numerous large (>2 mm) follicles. By 30 hr the ovulatory success (OS) was 3
%, at 36 hr post-LH, it had risen to 51%, at 42 hr
the OS decreased slightly (but not significantly;
see below) to 44%, and by 48 hr it increased again
to 83% (Fig. 1). The OS varied significantly with
time post-LH (F = 26.25; P < 0.01) and there were
significant differences between all time periods
(Tukey HSD; P < 0.05) except between 24 and 30
hr post-LH and 36 and 42 hr post-LH. Ovaries of
animals killed at 36 hr post-LH not only contained
developing multiple corpora lutea, but also a crop
of large (>2 mm) follicles. By 48 hr post-LH, the
ovaries of possums contained only a few large fol-
Oocyte collection
Using a 25-G needle and a 1-ml syringe containing sterile PBS + 10 IU heparin (Sigma, Australia), the oviducts and uteri were separately
flushed and their contents collected so that sites
of oocyte recovery could be determined. Mucoid
layer thickness of oocytes was measured using an
eyepiece graticule calibrated against a stage scale
and assessed on a scale of (1) Thin, <50 µm; (2)
medium, 50–70 µm; and (3) thick, >70 µm.
The ovaries of all animals were examined using a low power stereomicroscope. Ovulation sites
and the number of unruptured follicles >2 mm
diameter remaining were counted. The diameters
Fig. 1. The ovulatory success of female brushtail possums
treated with PMSG/LH with time post-LH injection.
610
A.M. GLAZIER
licles (>2 mm) and numerous small (<2 mm) follicles, the rest of the ovaries comprising developing corpora lutea.
The total number of follicles promoted per possum (N + N1) at each time period did not vary
significantly (F = 0.91; P > 0.05), demonstrating
that possums in each group responded to PMSG
in a similar manner.
Site of oocyte recovery
Ova were first collected from one animal at 30
hr post-LH (Fig. 1). At 36 hr post-LH, ova were
found predominantly in the oviduct (26/34). All
those in the oviduct had thin or medium mucoid
layers (Table 1). The remainder had thick mucoid
layers and were flushed from the uteri. At 42 hr
post-LH, ova flushed from the oviducts had only
thin mucoid layers (6/14). The remaining flushed
ova were collected from the uteri, having acquired
thick mucoid layers. By 48 hr post-LH, all recovered ova (38/38) were found in the uteri, and the
majority had acquired thick mucoid layers (Table
1) and were degenerating. No further ovulations
were observed after 42 hr post-LH as no further
oocytes were recovered from the oviducts at 48 hr
post-LH.
DISCUSSION
Ovulation in the induced ovulated possums occurred between 30 hr and 48 hr post-LH, with
most ovulations occurring between 30–36 hr postLH. The nonsignificant decrease in OS at 42 hr
is unexplained, however it would be expected that
the ovulatory success with time would have been
cumulative, but the large variation in number of
ovulated ova in animals killed at 36 hr masks this
effect. The finding in the oviducts at 42 hr postLH only of ova with thin mucoid layers, indicative of recent ovulation and compared to the
presence of thin- and medium-coated ova at 36 hr
post-LH, suggests that two phases or waves of
ovulation may have occurred. First, the largest
follicles released their ova in response to LH, all
at about the same time. Then a second wave of
ovulations occurred when follicles were released
from growth suppression by ovulation of the initial follicle cohort (Kaneko et al., ’95; Molinia et
al., ’98a). It is possible that the 6-hr time period
between observations in this study missed the onset of the second wave of ovulations. At 42 hr postLH, ova from the earlier round of ovulations had
passed through the oviducts, entered the uteri,
acquired thick mucoid layers, and were beginning
to degenerate.
The mechanisms controlling follicular development in the possum are poorly understood. With
the exception of the tammar wallaby (Macropus
eugenii) little is known about hormonal control of
folliculogenesis in marsupial species (Hinds et al.,
’96). To optimise fully the time of recovery of ovulated ova or time of artificial insemination (AI) in
the possum (Molinia et al., ’98a), it may be necessary to determine when the follicles promoted by
PMSG acquire LH receptors.
In this study ova were found in the uteri as
early as 36 hr post-LH and, as no ovulations were
observed before 30 hr post-LH, oviductal transport may be much more rapid than previously reported (Rodger, ’91).
Using a PMSG/GnRH hormone regime, Rodger
and Mate (’88) reported that ovulation occurred
about 24 hr post-GnRH injection. The time of ovulation using PMSG/LH in this study was significantly later. In tammar wallabies, natural ovulation
occurs as early as 20 hr after the LH surge (Harder
et al., ’85), but a recent induced ovulation study in
the tammar reported that ovulation did not start
until 36 hr post-LH injection (Molinia et al., ’98b).
This delayed ovulation may be due, however, to the
use of porcine FSH in place of PMSG to promote
follicular growth (Molinia et al., ’98b). When wallabies were manipulated to effect laparoscopic artificial insemination after anaesthesia (Molinia et al.,
’98a), there was an increase in the time of onset of
TABLE 1. The site of recovery and thickness of mucoid layers surrounding recovered oocytes at varying times post-LH
injection in induced ovulated brushtail possums (Trichosurus vulpecula)
Hours
post-LH
30
36
42
48
a
b
Site of oocyte recovery
Mucoid thickness (% of total oocytes recovered)
No. of
oocytes (n)
Oviduct
Uterus
Thin
1
34
14
38
1
26
6
0
0
8
8
38
100a
29a
43a
0
Site of oocyte recovery is oviduct.
Site of oocyte recovery is uterus.
Medium
0
47a
0
10b
Thick
0
24b
5b
90b
TIME OF OVULATION IN THE POSSUM
ovulation (F.C. Molinia, unpublished observations),
an effect reported for other eutherian species
(Howard et al., ’92); thus it would be reasonable to
assume that application of similar methods to the
possum would further delay the onset of ovulation.
Both the time of ovulation and speed of oviduct
transport are likely to be under hormonal control
to ensure close synchrony with the mucoid secretion and deposition that has a key role both in
preventing supernumary sperm from penetrating
the ovum and in structural organization of the
developing zygote (Breed, ’96).
The clear definition of the time of onset of ovulation following induced ovulation in possums will
now permit more efficient oocyte harvesting for
IVF assay development and artificial insemination protocols (Molinia et al., ’98a).
LITERATURE CITED
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