Journal of Reproduction and Fertility (2000) 120, 59–64

Natural and artificial methods for inducing the luteal phase in the koala (Phascolarctos cinereus)

S. D. Johnston1, M. R. McGowan1, P. O’Callaghan2, R. Cox3 and V. Nicolson2

1School of Veterinary Science and Animal Production, The University of Queensland, 4072, Australia; 2Lone Pine Koala Sanctuary, Jesmond Road, Fig Tree Pocket, 4069, Australia; and 3Bioquest Ltd, North Ryde, 2113, Australia

An experiment was conducted in which female koalas were mated for different durations of intromission and ejaculation to confirm that the luteal phase of the oestrous cycle in koalas is induced by the physical act of mating. Results showed that induction of a luteal phase in the koala usually required a complete duration of penile thrusting behaviour from the male. It is proposed that induction of a luteal phase in koalas may involve a copuloceptive reflex, triggered by the thrusting of the male’s penis into the female’s urogenital sinus. Although interrupted mating in koalas may be used to induce a luteal phase in preparation for an artificial insemination programme, this study showed that there is a 12.5% probability that pregnancy will result from semen prematurely emitted by the teaser male. A dose of 250 iu hCG was administered intramuscularly to eight oestrous females to determine whether it was possible to induce a luteal phase artificially. In contrast to control females, which received sterile saline injections, all females injected with hCG showed a significant increase in progestogen concentration above that of basal values, indicating that a luteal phase had been induced successfully.

Reviewing most of these studies, Hinds et al. (1996) Introduction concluded that although follicular growth in marsupials To date, the main method of synchronizing and inducing was consistently stimulated by administration of eCG, the oestrus in marsupials experimentally has been via the optimal dose rate of this preparation was usually lower than removal of pouch young (Renfree and Tyndale-Biscoe, 1978). that for eutherian species, and except for S. crassicaudata, Although this method can be used in experimental studies successful ovulation also required single or multiple of common species, the protocol usually requires killing injections of GnRH approximately 2–3 days after initial eCG the pouch young, a situation counter-productive to the treatment. More recently, a protocol using pig FSH and LH implementation of assisted breeding programmes. has been used to induce multiple ovulation in the tammar Induction of oestrus and ovulation in marsupials, like that wallaby (Macropus eugenii) (Nickel et al., 1996; Jungnickel of eutherian species, might also be achieved pharma- et al., 1998; Molinia et al., 1998a,b) and brushtail possum cologically. Since the original studies of Nelson and White (Trichosurus vulpecula) (Fletcher et al., 1998; Molinia et al., (1941) and Nelson and Maxwell (1942) on the opossum 1998a). (Didelphis virginiana), and Smith and Godfrey (1970) on the Although most marsupials studied ovulate spontaneously fat-tailed dunnart (Sminthopsis crassicaudata), considerable (Tyndale-Biscoe and Renfree, 1987), the grey short-tailed progress has been made towards the development of opossum (Monodelphis domestica) (Fadem et al., 1982; Hinds et methods for pharmacological induction of oestrus and al., 1992) and the brush-tailed bettong (Bettongia penicillata) ovulation in marsupials (Hinds et al., 1996; Mate et al., 1998). (Hinds and Smith, 1992; Smith, 1992, 1994) may be notable The most commonly used hormonal preparations involve a exceptions. In both of these species, oestrus can be induced combination of equine chorionic gonadotrophin (eCG), by the presence of the male, but it is uncertain whether followed by a series of treatments with either GnRH, pig LH ovulation follows spontaneously or requires the physical act or hCG (Nelson and White, 1941; Nelson and Maxwell, 1942; of mating. For example, studies of the woolley opossum Harding, 1969; Smith and Godfrey, 1970; Fletcher, 1983; (Caluromys philander) have indicated that although the Renfree et al., 1988; Rodger and Mate, 1988; Hayman et al., introduction of a male or exposure to male pheromones 1990; Rodger et al., 1992, 1993; Molinia et al., 1995, 1998a,b; can enhance and synchronize oestrus, ovulation occurs Hinds et al., 1996). spontaneously (Perret and Ben M’Barek, 1991). Johnston et al. (2000) presented evidence to indicate that, Received 20 September 1999. in koalas, the luteal phase of the oestrous cycle is induced by

© 2000 Journals of Reproduction and Fertility Ltd 0022–4251/2000 Downloaded from Bioscientifica.com at 10/02/2021 04:52:12PM via free access 60 S. D. Johnston et al. mating. Although the koala is apparently the only marsupial subsequently ejaculates which lasts approximately 32 s. known to show this reproductive pattern, it has been well Female koalas were randomly allocated into four experimental studied in eutherian mammals. The main neuroendocrine groups to determine at what point during mating a luteal pathways of copulation-evoked ovulation in rabbits have phase was induced. In the first group of eight animals, the been summarized by Ramirez and Lin Soufi (1994) and male koala was allowed to mount the female, secure a neck are collectively termed the copuloceptive reflex arc. The bite and thrust his penis into her urogenital sinus for a copuloceptive reflex can be triggered either by physical duration of approximately half the thrusting period (20 stimulation of the reproductive tract or ‘short-circuited’ thrusts in 10 s). In the second group of eight females, each pharmacologically at the hypothalamus or adenohypophysis male was allowed to complete the thrusting behaviour and by using either exogenous GnRH or LH-like preparations, was then removed. Completion of penile thrusting was respectively. characteristically signified in most males by two particularly On the basis of the hypothesis that ovulation in koalas also vigorous pelvic thrusts. Each of the six females in the third requires induction of a copuloceptive reflex, the present group was allowed to copulate with the male to a point study investigated whether techniques routinely used to representing half the ejaculatory period (16 s) as defined by induce ovulation in domestic cats and rabbits could also be O’Callaghan (1996). For groups 1–3, all females were mated used to induce a luteal phase in koalas. within 4 days of the onset of oestrus. Data from a final group Observations of the copulatory behaviour of koalas of ten females in which mating was not interrupted were (Smith, 1980; Johnston et al., 1997) were used in the first obtained from information presented in Johnston et al. (2000). experiment to determine whether it was possible to induce Females in this group were mated within 7 days of the onset ovulation by interrupting natural mating at the point at of oestrus. which the copuloceptive reflex arc had been sufficiently The presence of a copulatory plug or spermatozoa in the stimulated but sperm deposition into the female had not yet reproductive tract of the female after mating was determined occurred. The second experiment investigated whether by swabbing the cranial region of the urogenital sinus with a administration of hCG could be used to induce a luteal phase cotton wool swab and examining a smear preparation with a pharmacologically. phase-contrast microscope using a ϫ 40 objective. Blood samples (2 ml) for determination of progestogen concentration were collected from the cephalic vein of each koala on the day of mating (day 0) and 14 days later. Materials and Methods Johnston et al. (2000) showed that plasma progesterone concentrations of mated koalas at 14 days after mating Animals were significantly higher than those in unmated koalas. Sexually mature male (n = 28) and female koalas (n = 37) The details of the methodology of blood collection and from Lone Pine Koala Sanctuary, Brisbane were used in two progestogen assay procedures are described by Johnston experiments conducted from October 1996 to February 1998. et al. (2000). The collective term progestogen is used in this Only one female (F37) of the 65 koalas used in this study study because of the observed relatively high crossreactivity showed clinical evidence of illness (idiopathic respiratory (45%) with 5β-dihydroprogesterone (45%) (Johnston et al., disease) during the study; all others remained clinically 2000). The day after mating, each female was exposed to a normal. Housing and husbandry of koalas at Lone Pine ‘teaser’ male to determine whether she was still in oestrus. Koala Sanctuary have been described by Blaushard (1994). Detection of oestrus continued once a day until a birth or All procedures described in this study had the approval of subsequent oestrus was observed. the University of Queensland and Lone Pine Koala Sanctuary animal ethics committees.

Experiment 2: use of hCG for the induction of a luteal phase Detection of oestrus Ten female koalas were randomly allocated into two Throughout the breeding season, female koalas were experimental groups. The first (control) group consisted of ‘teased’ each day with a male to determine their reproduc- four oestrous females (days 1–3 after the onset of oestrus) tive status. Oestrous detection procedures and oestrous which received 0.83 ml sterile saline by injection into the behaviours have been described by Blanshard (1994) and in right supra-spinatus muscle. Sterile saline was injected using this volume by Johnston et al. (2000). As females came into a 25 gauge needle attached to 1 ml syringe. The treatment oestrus throughout the breeding season, they were randomly group consisted of six oestrous females (days 1–3 after onset allocated into the two experiments described below. of oestrus) which received 250 iu hCG (Chorulon®, Intervet, Castle-Hill) in 0.83 ml sterile saline by i.m. injection. Blood samples (2 ml) for determination of progestogen concentration were collected on the day of injection (day 0) Experiment 1: effect of amount of copulatory stimulation on and on day 14 and day 28 after injection of sterile saline or induction of a luteal phase hCG. When possible, oestrous behaviour was evaluated on During mating in koalas, O’Callaghan (1996) observed the day after injection and once a day until a subsequent that the male thrusts an average of 40 times in 20 s, and oestrus was detected.

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Confirmation of a luteal phase and statistical analysis In all eight females in which the duration of penile thrusting of the male was completed, the progestogen Induction of a luteal phase was confirmed by either birth concentrations on day 14 were significantly increased of an offspring or by a significantly high progestogen (6.8 Ϯ 1.6 ng ml–1). The mean (Ϯ SEM) number of thrusts and concentration 14 days after mating or injection of hCG. The duration of thrusting were 42.8 Ϯ 1.9 and 22.6 Ϯ 1.0 s, upper critical threshold value for the basal progestogen respectively. Although only one of these females gave birth, concentration was calculated by determining the upper limit the mean (Ϯ SEM) interoestrous interval of the remaining of a 99.95% confidence interval on the basis of a population females from which data were collected (n = 5) was (n = 41) of progestogen concentrations in blood samples 50.0 Ϯ 1.3 days, confirming that a luteal phase had been taken during oestrus in Expts 1 and 2. A progestogen induced. Although spermatozoa were found in the concentration greater than the threshold value was urogenital sinus of four of the females after complete considered to be indicative of a luteal phase. duration of penile thrusting, only one pregnancy resulted. As reported by Johnston et al. (2000), oestrus occurs Four of the eight females in this group continued to display 49.5 Ϯ 1.0 days after mating in non-parturient koalas, so that oestrus on the day after mating. females returning to oestrus around this period 50 days after The six females that received a complete duration of mating (Expt 1) or hCG injection (Expt 2) were considered to penile thrusting and half the duration of normal ejaculation have experienced a luteal phase. In Expt 2, an additional all became pregnant and gave birth. These females also blood sample was taken at 28 days after injection to had significantly increased concentrations of progestogen determine whether progestogen secretion was maintained (8.6 Ϯ 2.0 ng ml–1) on day 14 after mating. The mean (Ϯ SEM) throughout the luteal phase. number of thrusts and duration of thrusting were 41.7 Ϯ 1.6 Chi-squared analysis (Feldman and Gagnon, 1991) was and 29.5 Ϯ 2.5 s, respectively. Five of the six females showed used to compare the frequency of induction of a luteal phase no evidence of oestrous behaviour on the day after mating. for each amount of copulatory stimulation in Expt 1. Chi- In the group of ten females in which mating was not squared analysis was also used to compare the frequency of interrupted, six gave birth and all females had significantly induction of a luteal phase for koalas receiving either a sterile increased concentrations of progestogen (5.9 Ϯ 1.3 ng ml–1) saline injection or 250 iu hCG. An unpaired t test was used to on day 14 after mating. The four females that did not give compare the progestogen concentrations in pregnant koalas birth returned to oestrus; the mean (Ϯ SEM) interoestrous (day 14 after mating) in Expt 1 with concentrations in interval in these females was 49.0 Ϯ 1.4 days. The mean females induced to ovulate with 250 iu hCG (day 14 after (Ϯ SEM) number of thrusts and duration of thrusting for six of injection) in Expt 2. the animals was 43.0 Ϯ 3.6 and 24.2 Ϯ 1.0 s, respectively. Copulatory behaviour was not observed directly in the other four matings. The mean (Ϯ SEM) duration of ejaculation for the six observed matings was 27.3 Ϯ 4.2 s. None of the Results females in this group showed oestrous behaviour on the day after mating. Experiment 1 The proportion of koalas in which a luteal phase was The mean (Ϯ SEM) progestogen concentration from induced and in which pregnancy ensued as a result of 41 oestrous blood samples from Expts 1 and 2 was the various mating treatments is summarized (Table 1). 0.7 Ϯ 0.1 ng ml–1. The upper critical threshold value Chi-squared analysis revealed that successful induction of a indicative of successful induction of a luteal phase was luteal phase (χ2 = 22.2; P < 0.01) and birth (χ2 = 27.6; P < 0.01) estimated to be 1.3 ng ml–1. One outlier progesterone value were both dependent on the duration of mating stimulus. from Expt 2 (F34; 32.6 ng ml–1) was removed from the calculation of the mean. In six of eight females that were mated to a point Experiment 2 approximately half the normal duration of penile thrusting, the progestogen concentrations on day 14 were not The effect of administration of hCG during oestrus is significantly increased (0.9 Ϯ 0.2 ng ml–1), evidence that a summarized (Table 2). In three females that received an luteal phase had not been induced. The mean (Ϯ SEM) injection of sterile saline during oestrus, there was no interoestrous interval of these koalas was 30.4 Ϯ 2.0 days evidence of induction of a luteal phase: the progestogen (n = 5). However, two koalas in this group did show concentrations at day 14 (0.3 Ϯ 0.1 ng ml–1) and day 28 (0.5 Ϯ significantly increased concentrations of progestogen on day 0.3 ng ml–1) after injection were not significantly increased. 14. One of these females went on to give birth (progestogen However, the progestogen concentrations of the remaining concentration: 8.2 ng ml–1) and the other had an extended female (F34) were significantly increased both during oestrus cycle duration of 50 days (progestogen concentration: (day 0) and 28 days later. The progestogen concentration of 2.4 ng ml–1), indicating that in both cases a luteal phase had this koala decreased below the threshold concentration at been induced. All koalas in this group, except for the koala day 14 after mating. The mean (Ϯ SEM) interoestrous interval showing the 50 day oestrous cycle, showed evidence of of the four koalas was 28.3 Ϯ 1.7 days and all displayed oestrus the day after mating. In two animals, spermatozoa oestrus the day after the injection. were recovered from the urogenital sinus and one of these In contrast to the control animals, all six females injected gave birth to a normal offspring. with 250 iu hCG concentrations of progestogen were

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Table 1. Proportion of koalas in which a luteal phase was induced and in which pregnancy resulted after different amounts of copulatory stimulation

Amount of copulatory Females in which a luteal Females in which a stimulation phase was induced pregnancy resulted

Half duration of thrusting 2/8 1/8 Complete duration of thrusting 8/8 1/8 Half duration of ejaculation 6/6 6/6 Complete ejaculation 10/10 6/10

Table 2. The effect of an intramuscular injection of hCG (250 iu) during oestrus in the koala

Progestogen (ng ml–1) Female showed oestrus Female Day 0 Day 14 Day 28 Day 58 the day after hCG injection

Sterile saline F13 0.1 0.1 0.1 – Yes F33 0.9 0.3 1.1 – Yes F34 32.6 0.5 7.2 – Yes F46 0.4 0.2 0.3 – Yes

250 iu Chorulon F3 0.2 4.5 10.2 0.2 Yes F9 0.1 6.1 11.5 NA Yes F37 0.1 12.3 7.5 0.1 No F38 0.2 5.5 8.4 0 NA F39 0.2 10.0 42.7 0.1 NA F47 0.1 6.2 8.7 NA Yes

NA: not available. significantly (χ2 = 12.0; P < 0.01) above the threshold value at progestogen concentration of the pregnant females day 14 (7.4 Ϯ 1.2 ng ml–1) and day 28 (14.8 Ϯ 5.6 ng ml–1) examined in Expt 1 (7.6 Ϯ 1.2 ng ml–1; n = 14). after injection, indicating that a luteal phase had been induced. Observations for signs of oestrus on the day after hCG Discussion injection were only possible for four of the six females. Of these four females, only one koala failed to display oestrus Results from Expt 1 confirm the findings of Johnston et al. on the day after hCG injection. A subsequent return to (2000), that the luteal phase of the oestrous cycle in koalas is oestrus after hCG injection was observed in only one of six induced by the physical act of mating and typically requires females. This female returned to oestrus at day 54 after hCG a full duration of penile thrusting from the male. injection. Unfortunately, as the study site was experiencing Interestingly, there also appears to be some significant higher than normal temperatures during the period in which individual variability in the amount of stimulation required the four females injected with hCG were expected to return to induce a luteal phase. After only 10s of penile thrusting to oestrus (February–March 1998) it was not possible to one female subsequently had increased progestogen determine whether failure to show oestrus in these females concentrations, which are indicative of a luteal phase, and represented a failure of hCG to stimulate a normal luteal another gave birth to a normal offspring. However, exposure phase, or was associated with heat stress. A fourth blood (without direct contact) to males displaying typical pre- sample was taken at day 58 after the original hCG injection to mating behaviour (urination, bellowing, scent marking) does determine whether these four females were experiencing not result in the induction of a luteal phase (Johnston et al., a prolonged luteal phase. At day 58, the progestogen 2000). There was also considerable individual variability concentration of all females had decreased below the with respect to the timing of emission of the sperm rich threshold value (0.1 Ϯ 0.04 ng ml–1), indicating that the luteal fraction. Although spermatozoa were not usually present in phase had ended. The same four females ultimately the urogenital sinus of the female after 10 s of penile displayed at least one further oestrus before the end of the thrusting by the male, two males ejaculated spermatozoa breeding season. relatively early in their thrusting periods. In one of these The mean (Ϯ SEM) progestogen concentration at day 14 in males a sufficient number of spermatozoa was released females injected with 250 iu hCG (7.4 Ϯ 1.2 ng ml–1; n =6) during the first 10 s of thrusting for conception to occur. was not significantly different from the mean (Ϯ SEM) On completion of thrusting, four of eight males had

Downloaded from Bioscientifica.com at 10/02/2021 04:52:12PM via free access Induction of a luteal phase in the koala 63 ejaculated some spermatozoa into the urogenital sinus of the explained by an error in oestrous detection or it may be an female. However, as only one of the eight females became example of non-oestrous expression of behavioural oestrus. pregnant, it appears that in most cases only a relatively small Reports of spontaneous ovulation in induced ovulators are number of spermatozoa are emitted by completion of not uncommon (Flowerdew, 1987). thrusting behaviour (compared with the full copulatory The data reported in Expts 1 and 2 support the conclusion sequence). All six females that were mated through to the that a luteal phase and most probably ovulation were midpoint of ejaculation subsequently gave birth, indicating successfully induced after both interrupted mating and that spermatozoa required for successful fertilization may be administration of 250 iu hCG. However, Johnston et al. (2000) ejaculated relatively early during the so called ejaculatory showed that the koala ovary contains a high proportion of period (Smith, 1980). These results also demonstrate that the luteinized interstitial tissue, apparently formed from atretic original definitions of thrusting and the ejaculatory period in follicles. Although improbable, it needs to be acknowledged koalas given by Smith (1980) are arbitrary, as most male that the occurrence of a luteal phase induced by either koalas in the present study ejaculated plug material, and in a interrupted mating or hCG may be the result of progestogen minority of cases, a sperm rich fraction, during the period of production from luteinized atretic follicles. Further direct penile thrusting. histological observations of koala ovaries are required to Results from the present study indicate that interrupted confirm that an increase in progestogen concentration mating could be used as a means of inducing the luteal phase equivalent to that of a luteal phase is actually associated with in female koalas in preparation for artificial insemination. ovulation. However, as there is also a 12.5% probability that pregnancy may result from semen prematurely emitted by the ‘teaser’ The authors would like to thank Tom Sakurabu and Toshi male, the parentage of any resulting offspring must be verified. Akiyama previous General Managers of Lone Pine Koala Sanctuary Results from Expt 2 indicate that an i.m. injection of 250 iu (LKPS) for allowing access to their facilities and collaboration with hCG at the time of oestrus results in the induction of a luteal their staff. In particular, Jacque Brumm and Karen Neilson who phase in koalas. Although it was not possible to determine helped in oestrus detection procedures and collecting koala blood samples. They would also like to acknowledge the generous support directly whether ovulation was also induced, the progestogen of Jim Kelly of Bioquest Ltd, (North Ryde, Sydney) for providing the concentrations of koalas (at day 14 after injection with hCG) laboratory space and equipment to run hormone assays. The authors were not significantly different from those of pregnant are grateful to F. N. Carrick for providing a critical review of the animals at a similar stage in the luteal phase. These results manuscript. The work presented in this paper was supported by compare with the use of hCG (in combination with eCG) research grants supplied by Lone Pine Koala Sanctuary and the in other marsupials to induce ovulation in D. virginiana University of Queensland. (Nelson and White, 1941; Nelson and Maxwell, 1942) and S. crassicaudata (Smith and Godfrey, 1970), but no offspring were produced in these species. Harding (1969) attempted to References use a similar combination of hormones to induce ovulation in T. vulpecula, but most of the oocytes became trapped in Blanshard W (1994) Medicine and husbandry of koalas. In Wildlife, Proceedings luteinized follicles. Fletcher (1983) also used 500 iu hCG to Number 223 pp 547–623. University of Sydney Post Graduate Committee in Veterinary Science, Sydney produce luteal conditions in the kowari (Dasyuroides byrnei). Fadem BH, Trupin GL, Maliniak E, Vanderberg JL and Hayssen V (1982) In the present study, the occurrence of oestrus on the day Care and breeding of the gray, short-tailed opossum (Monodelphis domestica) after mating decreased with an increasing amount of Laboratory Animal Science 32 405–409 copulatory stimulation. 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