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 WhenuaLandcare 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. 283:608611, 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: email@example.com 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 Breed WG. 1996. Egg maturation and fertilization in marsupials. Reprod Fertil Dev 8:617–643. 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