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Hormonal control of birth behavior in the bandicoot (Perameles gunnii: Marsupialia) and other marsupials
Physiology & Behavior 72 (2001) 527 ± 532
Hormonal control of birth behavior in the bandicoot (Perameles gunnii: Marsupialia) and other marsupials Tracey Smith, Alan MacFadyen, Randy Rose* School of Zoology, University of Tasmania, GPO Box 252-05, Hobart, Tasmania 7001, Australia Received 9 December 1999; received in revised form 2 October 2000; accepted 3 November 2000
Abstract The barred bandicoot is a small marsupial, locally common on the island of Tasmania, Australia. Several marsupial species have been shown to respond to injections of prostaglandin F2a or oxytocin by demonstrating birth behavior but that if an inhibitor of prostaglandin synthesis was given prior to oxytocin, no birth behavior was demonstrated. The barred bandicoot has behaved in a similar manner, demonstrating a significant increase in grooming and three other behaviors (crouching/prone, lateral and `birth') were only seen after injection of PGF2a or oxytocin. The fact that after injection of oxytocin there was an increase in the latency till a birth response confirms a previous finding in another species. Subsequently, we have repeated these experiments with other marsupial groups, with similar results, which suggest that prostaglandin F2a stimulates birth behavior and oxytocin stimulates prostaglandin production in most marsupials. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Birth behavior; Marsupials
1. Introduction Birth behavior in marsupials is of considerable interest because despite their small size, young crawl unaided to the mother's pouch attaching to one of the teats therein. Behavior at birth was best described in the red kangaroo (Macropus rufus) [22] and more recently in the tammar wallaby (Macropus eugenii) [17 ±19]. These authors describe very similar events in both species: Initially, there is a period of pouch cleaning, which increases in intensity, the mother then adopts a birth posture characteristic for that group of marsupials (in macropods, this is usually propped up against a tree with the tail passed forward between the hindlimbs). Licking of the uro-genital area increases and become vigorous during which time the mother appears oblivious to external stimuli; birth then occurs and the young move unaided to the pouch. The mother does not lick or prepare a pathway for the young. There is far less information available on birth behavior in other Australian marsupials although the birth of a bandicoot (Isoodon macrourus) has been described in some detail * Corresponding author. Tel.: +61-3-6226-2633 (office); fax: +61-36226-2745; http://www.utas.edu.au/docs/zoology/rose.html. E-mail address: [email protected] (R. Rose).
[7,11]. In this species, rather than adopting a propped up position, the bandicoot lies on its side with one leg raised and licks vigorously at her uro-genital sinus. Again, the animal appeared oblivious to external stimuli such as flashlights [11]. While it was obvious that such behaviors were hormonally induced it was not until later work [9,23] that it first became clear that prostaglandins were involved. The levels of prostaglandin F2a increase at birth [10,17], and it was shown that injections of PGF2a could induce birth behavior in both nonpregnant female and male tammar wallabies [23]. Subsequently, we have shown that PGF2a could induce birth behavior in other species of marsupial including the brush-tail possum (Trichosurus vulpecula) and the shortnosed bandicoot (I. macrourus) [6]. More recently, we showed that not only would PGF2a induce birth behavior in females, males and pouch young of the Tasmanian bettong (Bettongia gaimardi, a small rat ±kangaroo found only on the island of Tasmania) but that oxytocin also could induce the same behavior [20], though the latency of time until the appearance of birth behavior was increased. We also showed that if oxytocin was given with a prostaglandin synthesis inhibitor, birth behavior did not occur. From this, we concluded that in this species, oxytocin induced birth behavior via the synthesis of prostaglandin. Oxytocin (some
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marsupials produce mesotocin [2]) is released at birth in the tammar wallaby [13]. We wished to know whether the responses to both prostaglandin and oxytocin were only found in the superfamily Macropodoidea (kangaroos, wallabies and rat ± kangaroos) or were present in other marsupial families. Hence, we initiated a pilot study to test a range of Tasmanian marsupials from a number of families. The barred bandicoot (Perameles gunnii) is a small bandicoot (Peramelidae) that while relatively common in Tasmania, elsewhere in Australia, it is an endangered species. As birth behavior in this species has never been described, we wished to make a detailed behavioral study of its hormonal induction. Male and female barred bandicoots were injected with prostaglandin or oxytocin and detailed observations were made of the appearance of birth behavior.
ing behavior (0.1 ml was administered 30 min before oxytocin (0.3 ml) to allow the former compound to take effect). Individuals were placed in the arena approximately 1 h before a trial to allow the animal to become accustomed to the environment. During this time, the animals were checked regularly. They were then removed briefly from the arena injected with the required substance and returned to the arena. All this was achieved as quickly as possible ( < 60 s).
2. Methods and materials
All injections (except for oxytocin) were in a volume of 0.1 ml: saline (0.9%), prostaglandin F2a (0.025 mg) and Finadyne (5 mg). Before the oxytocin experiment could start, a dose ±response experiment was undergone in order to determine what dose was required for birth behavior to be exhibited: Full birth behavior did not appear until a dosage was given of 0.3 ml oxytocin (10 IU ml ÿ 1).
2.1. Birth behavior As this behavior has not been previously described, we used the following description of birth from Ref. [11] for the related bandicoot, I. macrourus as a guide: Immediately before birth the expectant mother assumed the birth position. She was on her side. With one leg raised, steadying herself with her front feet on the floor of the cage, and her head twisted so that she was facing her rear end. Vigorous licking of the urogenital opening preceded the sudden emergence of two young. They appeared to be born free of membranes but they had intact allantoic stalks.
An observation arena made of glass measuring 1 1 0.6 m fitted with a wire lid. A piece of carpet was placed in the bottom of the arena; black plastic was used to black out the back panel and the lid to prevent light penetration (bandicoots are nocturnal). Six barred bandicoots were captured in bushland 25 km from the University of Tasmania and housed in the animal holding area. They were fed cooked mincemeat, sunflower seeds, apples and earthworms on occasions. They were held for at least 1 month before experiments started. Six mature individuals were involved in all the experiments, three males; M3, M4 and M9 (1003.3 41.6 g) and three nonpregnant females without pouch young; F1, F5 and F7 (806.7 30.6 g). Saline (sodium chloride injection BP 0.09% 100 ml, David Bull Laboratories, DBL) was used as a control; prostaglandin F2a (Lutalyse 5 mg/ml, UpJohn); and oxytocin (Syntocinon, which contained no vasopressin, 10 IU/ml, Sandoz Pharma). If a result was obtained with both prostaglandin and oxytocin, a prostaglandin synthesis inhibitor (Flunixin meglumine 50 mg/ml Finadyne, Heriot Agvet, Schering, USA) was then administered with oxytocin to determine which of the hormones was responsible for birth-
2.2. Site of injection Individuals were injected intramuscularly into the thigh muscle. Where more than one injection was required to be administered, the injection was alternated between thigh muscles. 2.3. Dosage rates
2.4. Method of recording Preliminary observations of the behavioral components involved with the birthing procedure were noted to enable easy identification of each behavior during the trials. During the experiments, as much behavioral information as possible was recorded including the frequencies, descriptions and durations of behavior. 2.5. Statistical analysis Two-factor ANOVAs were performed for all recorded behaviors with treatment and gender factors. Latency to birth behavior was analysed using the Student's t test. 2.6. Pilot study of other marsupial species A number of marsupial species housed at the University of Tasmania were available for testing (Table 1). A standard dose of PGF2a (0.025 mg kg ÿ 1) or oxytocin (0.5 ± 1 IU) was given intramuscularly in the rump and animals watched for the appearance of birth behavior for the following hour. As this wasapilotstudy,lessbehavioralobservationswereundertaken. 3. Results 3.1. Behavioral descriptions from Per. gunnii Some of the behaviors described here are not solely associated with the birthing procedure but are maintenance
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3.3. Investigative behavior
Table 1 Tasmanian marsupial species used in the pilot study on birth behavior Family
Common name
Scientific name
N
Dasyuridae Dasyuridae Peramelidae Petauridae Phalangeridae Macropodidae Potoroidae Macropodidae
Spotted-tailed quoll Tasmanian devil Southern Brown bandicoot Sugar glider Brushtail possum Tasmanian pademelon Long-nosed potoroo Bennett's wallaby
Dasyurus maculatus Sarcophilus harrissi Isoodon obesulus Petaurus breviceps Tr. vulpecula Thylogale billardierii Potorous tridactylus Macropus rufogriseus
1 4 3 5 4 4 4 2
This is a general behavior and involved investigation of the glass observation arena. Usually, the individual moved around the area sniffing and pawing at the glass, the carpet and the straw on the base. Individuals would sometimes spend large amounts of time in the corners attempting to pull up the carpet. 3.4. Grooming
behaviors. When associated with birth behavior some are performed with increased frequency. These behaviors have not been reported for this species, Per. gunnii, or for any other bandicoot species apart from an actual observation of birth in I. macrourus in captivity [7,11].
This is a normal, maintenance behavior but intensifies and is performed more often when associated with birth. The most common parts of the body that are groomed are, in descending order; the hind, the forepaws, hind flank and face groom. Ear scratching is also often associated with face grooming.
3.2. Sitting and standing This general behavior involves the individual either sitting or standing still for a period of time. Sometimes, the animal may be crouched down as if asleep, at other times, it may be standing on its hind legs with its nose in the air and forefeet poised. Another common position is with the individual on all four feet but one forefoot is raised and the snout is pointed forwards.
3.5. Crouching/prone Crouching involves simply squatting down. However, the prone position involves the animal stretched out with its forefeet out in front and the nose resting on the ground or on the forepaws. This position is an indicator and a forerunner leading to the birth position.
Fig. 1. Photograph of `birth' behavior in a male bandicoot after injection with oxytocin. Note that the scrotum is anterior to the penis in marsupials.
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groomed and licked; in males, the area near the uro-genital sinus and the testes was licked (Fig. 1). During the trials and after injection of prostaglandin or oxytocin, individuals were completely preoccupied in performing the various behaviors and were not distracted by external stimuli. Two-factor ANOVAs for all recorded behaviors gave the following results: There were no significant differences between the sexes in their responses to any of the treatments so their data were pooled. Significant differences in response to treatments did occur for Groom [ F(3,1) = 11.04, P = .0004]; crouching/prone [ F(3,1) = 17.84, P = .0001]; lateral [ F(3,1) = 8.72, P = .0012] and `birth' [ F(3,1) = 3.26, P = .04]. Only the prostaglandin and oxytocin (without Finadyne) treated groups demonstrated the crouching/prone, lateral and `birth' behaviors. Although all groups groomed this was significantly greater in the groups treated with prostaglandin and oxytocin (without Finadyne) (Fig. 2). Fig. 2. Occurrence of behaviors (per 60 min) (mean S.E.) in Per. gunnii (three males and three females) after treatment with saline, prostaglandin, oxytocin and Finadyne followed by oxytocin. Numbers refer to total responding if not all six.
4. The behavioral sequence preceding `Birth' The initial behaviors exhibited when the trial first begins are investigative, sitting and standing. These behaviors appear to last from the beginning until approximately 15 ± 20 min into the trial. Grooming becomes evident at about 13± 14 min and continues for 35 min in males and 20 min in females. There is a slight difference between males and females in relation to the timing of the onset of prone position. Males appear to adopt the prone posture at a later time than females (15 and 10 min, respectively) although this difference is not significant and is possibly the result of the large amount of individual variation encountered throughout the experimental regime. Lateral position occurs at approximately the same time for both sexes (15 min) while birth position and birthing behavior begins at 20 min. All of these behaviors were exhibited in a cyclic fashion and did not occur simultaneously over the whole period.
3.6. Lateral Here, the individual lies on its side, either right lateral or left lateral. This is similar to the normal sleeping posture but differs in that when asleep, they curl up into a tight ball but in the `lateral position' the individual stretches out and does not curl up at all. Grooming of the forepaws, face and hind legs may also be performed while in this position. 3.7. `Birth' The birth position is the same as that adopted for the lateral but the top hind leg is abducted (lifted out of the way) and the area surrounding and leading to the pouch is
Table 2 Pilot study of the effects of PGF2a and oxytocin on Marsupials (includes all previously published data) Family
Common name
Scientific name
N PGF2aa Oxytocina Reference
Macropodidae Potoroidae Macropodidae Potoroidae Macropodidae Phalangeridae Petauridae Peramelidae Peramelidae Dasyuridae Dasyuridae Didelphidaeb
Tammar wallaby Tasmanian bettong Tasmanian pademelon Potoroo Bennett's wallaby Brushtail possum Sugar glider Brown bandicoot Barred bandicoot Tasmanian devil Spotted-tailed quoll Grey opossum
Ma. eugenii B. gaimardi Th. billardierii Po. tridactylus M. rufogriseus Tr. vulpecula Pet. breviceps I. obesulus Per. gunnii S. harrissi D. maculatus Monodelphis domestica
10 15 11 4 4 4 3 5 6 4 1 30
a b c
+ + + + + + + + + + +
ÿ + + + + + + + ÿ +c
[9] [23]; Shaw, 1989; Shaw, personal communication [20] Rose and MacFadyen, unpublished Rose and MacFadyen, unpublished Rose and MacFadyen, unpublished; Loudon and Shaw, unpublished [6]; Rose and MacFadyen, unpublished [20] [6]; Rose and MacFadyen, unpublished This study Rose and MacFadyen, unpublished; This study Rose and MacFadyen, unpublished [21]; Fadem, Rose and Dziadosz, unpublished
Minimal dose of PGF2a that produced an effect was 0.05mg. kg ÿ 1: all oxytocin doses were 1 IU kg ÿ 1. American species, all other are Australian. Males did not respond.
T. Smith et al. / Physiology & Behavior 72 (2001) 527±532
If these results are compared with the control (saline), it is evident that no birth or components of birth behavior are exhibited (Fig. 2). The responses after an injection of a 0.3 ml dose of oxytocin are displayed in Fig. 2. Not all individuals performed the whole birthing sequence, i.e. birth position; however, components of behaviors including `prone' and `lateral' were performed with regularity. Overall, the adoption of birth position was less frequent for both males and females compared with that exhibited in the PGF2a experiment. The components of birth behavior (crouching/prone, lateral and `birth') did not occur after injection of finadyne and oxytocin. There is no data for the control or Finadyne plus oxytocin treatments for latency (the time interval between injection and the adoption of the birth position) since birth behavior did not occur. Both male and female latencies are similar for the PGF2a trials (18.3 12.3 and 19.3 11.4 min, respectively) but this increased in the oxytocin experiments (32.2 14.2 and 45.4 10.5 min, respectively). There was no significant difference between the sexes for either treatment (PGF2a, P = .29) or (Oxy, P = .39). 4.1. Pilot study All species tested showed clear-cut birth behavior in response to prostaglandin and several did after oxytocin injection (Table 2). 5. Discussion These experiments showed that the barred bandicoot responds to PGF2a by exhibiting birth behavior as previously shown in the tammar wallaby, brush-tail possum and Tasmanian bettong. In fact, every marsupial species that we have tested in our laboratory responds to PGF2a with birth behavior [20]. In addition, we have shown recently that female South American opossums, Mo. domestica also show a birth response to prostaglandin and oxytocin, although males did not respond to oxytocin [21]. We also have shown that birth behavior in the bandicoot can be elicited by oxytocin. Our results suggest that oxytocin acts via the synthesis of PGF2a: We believe this because oxytocin had no effect after prior treatment with Finadyne an inhibitor of PGF2a synthesis. Oxytocin is well known to stimulate the synthesis of PGF2a in many species [4,5]. This would explain the longer latency between injection of oxytocin and the behavioral changes. It was found that the prostaglandin metabolite PGFM was elevated within 5 min after an injection of oxytocin in the red deer Cervus elaphus [5]: In our experiments, the latency increased by approximately 15 ±20 min. The source of the synthesised prostaglandin is unclear; if it is from reproductive tissue, it is surprising that in the Tasmanian bettong, B. gaimardi immature females, pouch young and males respond as quickly as mature females [20]. The role of oxytocin and
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prostaglandins in male marsupials is unclear. However, oxytocin/mesotocin has been found in the testis of the bandicoot I. macrourus [1] and its receptor gene in the prostate of the tammar wallaby [14]. Why male marsupials should respond to prostaglandin and oxytocin with female behavior (birth) is surprising. Perhaps this behavior is `hardwired' in the brain and not easily acted upon by natural selection. Alternatively, the gene(s) responsible may be closely associated with other genes essential for both sexes. The natural levels of prostaglandin and oxytocin might never be sufficiently high to result in the behavior being expressed in males. The role of prostaglandins in the activation of birth behavior in eutherians has been little studied though some eutherian species (e.g. pig, rat and hamster) demonstrate parturient behavior in response to prostaglandin [3,8]: It is also suggested to be an ancient pathway found in some lower vertebrates (fish, amphibia and reptiles) [8]. Oxytocin is known to stimulate maternal behavior in some eutherian mammals [15,16]. It is of great interest that all major groups of Australian marsupials and the one South American form so far tested [21] respond to prostaglandin and oxytocin in a similar way. This suggests that this behavior may have been present in the common ancestor to both groups. One practical application of these results is in the ability to simulate birth in species where a description of such behavior is hitherto lacking. For example, when our experiments were undertaken with Tasmanian devils (S. harrissi) no information on its birth behavior was available. Subsequently, such information has come to light in a film `The Devil's Playground' [12] of a Tasmanian devil giving birth corroborating our results. The finding that prostaglandin precipitates birth behavior [6,20,21,23] has been reproduced in the present study. The finding that oxytocin has a similar effect, only previously shown in the bettong is likely to lead to new areas of research into the relationships between marsupial endocrinology and behavior. For example, the fact that males, females and juvenile marsupials respond in a similar manner suggests that there is an area of the brain in all marsupials that elicits maternal (birth) behavior. The location of this region warrants further study as does the interaction of prostaglandin and oxytocin at birth. Acknowledgments We thank Dr. L. Barmuta for his advice with the statistical analysis and Dr. Sue Jones for her assistance with the experiment. References [1] Bathgate RA, Sernia C, Gemmell RT. Arginine vasopressin- and oxytocin-like peptides in the testis of two Australian marsupials. Peptides 1993;14:701 ± 5.
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[2] Bathgate RA, Parry LJ, Fletcher TP, Shaw G, Renfree MB, Gemmell RT, Sernia C. Comparative aspects of oxytocin-like hormones in marsupials. Adv Exp Med Biol 1995;395:639 ± 55. [3] Blackshaw JK, Blackshaw AW. The effects of prostaglandin PGF2a on the behaviour of the domestic non-pregnant sow and boar. Anim Prod 1982;14:550 ± 2. [4] Flint APF, Sheldrick EL. Ovarian oxytocin and the maternal recognition of pregnancy. J Reprod Fertil 1986;76:831 ± 9. [5] Flint APF, Jabbour HN, Loudon ASI. Oxytocin stimulates uterine prostaglandin F2a in red deer Cervus elaphus. Reprod Fertil Dev 1994;6:269 ± 71. [6] Gemmell RT, MacFadyen AS, Rose RW. The induction of parturient behaviour in possums and bandicoots. Aust Mamm 1991;14:133 ± 6. [7] Gemmell RT, Veith C, Nelson J. Birth in the northern brown bandicoot. Isoodon macrourus (Marsupialia: Peramelidae). Aust J Zool 1999;47:517 ± 28. [8] Guillette LJ, Dubois DH, Cree A. Prostaglandins, oviducal function, and parturient behavior in non-mammalian vertebrates. Am J Physiol 1991;260:R854 ± 61. [9] Hinds LA, Tyndale-Biscoe CH, Shaw G, Fletcher TP, Renfree MB. Effects of prostaglandin and prolactin on luteolysis and parturient behaviour in the non-pregnant tammar, Macropus eugenii. J Reprod Fertil 1990;88:323 ± 33. [10] Lewis PR, Fletcher TP, Renfree MB. Prostaglandin in the peripheral plasma of tammar wallabies at parturition. J Endocrinol 1986;111: 103 ± 9. [11] Lyne AG. Gestation and birth in the marsupial Isoodon macrourus. Aust J Zool 1974;22:303 ± 9. [12] Morris R. The Devil's Playground. Natural History New Zealand Dunedin, NZ. [13] Parry LJ, Guymer F, Fletcher TP, Renfree MB. Release of an oxytocic peptide at parturition in a marsupial, Macropus eugenii. J Reprod Fertil 1996;107:191 ± 8.
[14] Parry LJ, Bathgate RA. Mesotocin receptor gene and protein expression in the prostate gland, but not the testis, of the tammar wallaby, Macropus eugenii. Biol Reprod 1998;59:1101 ± 7. [15] Pederson CA, Prange AJ. Induction of maternal behaviour in virgin female rats after intracerebroventricular administration of oxytocin. Proc Natl Acad Sci U S A 1979;76:6661 ± 5. [16] Pederson CA, Ascher JA, Monroe YL, Prange AJ. Oxytocin induces maternal behaviour in virgin female rats. Science 1982;216:648 ± 50. [17] Renfree MB, Shaw G, Fletcher TP. Evidence for the essential role of prostaglandins for parturition in a marsupial, Macropus eugenii. J Reprod Fertil 1994;102:433 ± 46. [18] Renfree MB, Fletcher TP, Blanden DR, Lewis PR, Shaw G, Gordon K, Short RV, Parer-Cook E, Parer D. Physiological and behavioural events around the time of birth in macropodid marsupials. In: Grigg G, Jarman P, Hume ID, editors. Kangaroos, wallabies and rat ± kangaroos. vol. 1, Sydney, Australia: Surrey Beatty and Sons, 1990; pp. 323 ± 37. [19] Renfree MB, Parry LJ, Fletcher TP, Shaw G. Control of parturition in the marsupial Macropus eugenii. In: Saunders NR, Hinds LA, editors. Recent advances in marsupial biology: University of NSW Press, Sydney, Australia, 1996. pp. 31 ± 40. [20] Rose RW, MacFadyen AS. Oxytocin and prostaglandin F2a induce birth behavior in the bettong Bettongia gaimardi. Horm Behav 1997; 31:120 ± 5. [21] Rose RW, Fadem BH. The hormonal control of birth behavior in the Gray short-tailed opossum (Monodelphis domestica). Horm Behav 2000;37:163 ± 7. [22] Sharman GB, Calaby JH. Reproductive behaviour in the red kangaroo, Megaleia rufa, in captivity. CSIRO Wildl Res 1964;9:58 ± 85. [23] Shaw G. The control of parturient behaviour by prostaglandin F2a in the tammar wallaby (Macropus eugenii). J Reprod Fertil 1990;88: 335 ± 42.
Hormonal control of birth behavior in the bandicoot (Perameles gunnii: Marsupialia) and other marsupials Tracey Smith, Alan MacFadyen, Randy Rose* School of Zoology, University of Tasmania, GPO Box 252-05, Hobart, Tasmania 7001, Australia Received 9 December 1999; received in revised form 2 October 2000; accepted 3 November 2000
Abstract The barred bandicoot is a small marsupial, locally common on the island of Tasmania, Australia. Several marsupial species have been shown to respond to injections of prostaglandin F2a or oxytocin by demonstrating birth behavior but that if an inhibitor of prostaglandin synthesis was given prior to oxytocin, no birth behavior was demonstrated. The barred bandicoot has behaved in a similar manner, demonstrating a significant increase in grooming and three other behaviors (crouching/prone, lateral and `birth') were only seen after injection of PGF2a or oxytocin. The fact that after injection of oxytocin there was an increase in the latency till a birth response confirms a previous finding in another species. Subsequently, we have repeated these experiments with other marsupial groups, with similar results, which suggest that prostaglandin F2a stimulates birth behavior and oxytocin stimulates prostaglandin production in most marsupials. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Birth behavior; Marsupials
1. Introduction Birth behavior in marsupials is of considerable interest because despite their small size, young crawl unaided to the mother's pouch attaching to one of the teats therein. Behavior at birth was best described in the red kangaroo (Macropus rufus) [22] and more recently in the tammar wallaby (Macropus eugenii) [17 ±19]. These authors describe very similar events in both species: Initially, there is a period of pouch cleaning, which increases in intensity, the mother then adopts a birth posture characteristic for that group of marsupials (in macropods, this is usually propped up against a tree with the tail passed forward between the hindlimbs). Licking of the uro-genital area increases and become vigorous during which time the mother appears oblivious to external stimuli; birth then occurs and the young move unaided to the pouch. The mother does not lick or prepare a pathway for the young. There is far less information available on birth behavior in other Australian marsupials although the birth of a bandicoot (Isoodon macrourus) has been described in some detail * Corresponding author. Tel.: +61-3-6226-2633 (office); fax: +61-36226-2745; http://www.utas.edu.au/docs/zoology/rose.html. E-mail address: [email protected] (R. Rose).
[7,11]. In this species, rather than adopting a propped up position, the bandicoot lies on its side with one leg raised and licks vigorously at her uro-genital sinus. Again, the animal appeared oblivious to external stimuli such as flashlights [11]. While it was obvious that such behaviors were hormonally induced it was not until later work [9,23] that it first became clear that prostaglandins were involved. The levels of prostaglandin F2a increase at birth [10,17], and it was shown that injections of PGF2a could induce birth behavior in both nonpregnant female and male tammar wallabies [23]. Subsequently, we have shown that PGF2a could induce birth behavior in other species of marsupial including the brush-tail possum (Trichosurus vulpecula) and the shortnosed bandicoot (I. macrourus) [6]. More recently, we showed that not only would PGF2a induce birth behavior in females, males and pouch young of the Tasmanian bettong (Bettongia gaimardi, a small rat ±kangaroo found only on the island of Tasmania) but that oxytocin also could induce the same behavior [20], though the latency of time until the appearance of birth behavior was increased. We also showed that if oxytocin was given with a prostaglandin synthesis inhibitor, birth behavior did not occur. From this, we concluded that in this species, oxytocin induced birth behavior via the synthesis of prostaglandin. Oxytocin (some
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T. Smith et al. / Physiology & Behavior 72 (2001) 527±532
marsupials produce mesotocin [2]) is released at birth in the tammar wallaby [13]. We wished to know whether the responses to both prostaglandin and oxytocin were only found in the superfamily Macropodoidea (kangaroos, wallabies and rat ± kangaroos) or were present in other marsupial families. Hence, we initiated a pilot study to test a range of Tasmanian marsupials from a number of families. The barred bandicoot (Perameles gunnii) is a small bandicoot (Peramelidae) that while relatively common in Tasmania, elsewhere in Australia, it is an endangered species. As birth behavior in this species has never been described, we wished to make a detailed behavioral study of its hormonal induction. Male and female barred bandicoots were injected with prostaglandin or oxytocin and detailed observations were made of the appearance of birth behavior.
ing behavior (0.1 ml was administered 30 min before oxytocin (0.3 ml) to allow the former compound to take effect). Individuals were placed in the arena approximately 1 h before a trial to allow the animal to become accustomed to the environment. During this time, the animals were checked regularly. They were then removed briefly from the arena injected with the required substance and returned to the arena. All this was achieved as quickly as possible ( < 60 s).
2. Methods and materials
All injections (except for oxytocin) were in a volume of 0.1 ml: saline (0.9%), prostaglandin F2a (0.025 mg) and Finadyne (5 mg). Before the oxytocin experiment could start, a dose ±response experiment was undergone in order to determine what dose was required for birth behavior to be exhibited: Full birth behavior did not appear until a dosage was given of 0.3 ml oxytocin (10 IU ml ÿ 1).
2.1. Birth behavior As this behavior has not been previously described, we used the following description of birth from Ref. [11] for the related bandicoot, I. macrourus as a guide: Immediately before birth the expectant mother assumed the birth position. She was on her side. With one leg raised, steadying herself with her front feet on the floor of the cage, and her head twisted so that she was facing her rear end. Vigorous licking of the urogenital opening preceded the sudden emergence of two young. They appeared to be born free of membranes but they had intact allantoic stalks.
An observation arena made of glass measuring 1 1 0.6 m fitted with a wire lid. A piece of carpet was placed in the bottom of the arena; black plastic was used to black out the back panel and the lid to prevent light penetration (bandicoots are nocturnal). Six barred bandicoots were captured in bushland 25 km from the University of Tasmania and housed in the animal holding area. They were fed cooked mincemeat, sunflower seeds, apples and earthworms on occasions. They were held for at least 1 month before experiments started. Six mature individuals were involved in all the experiments, three males; M3, M4 and M9 (1003.3 41.6 g) and three nonpregnant females without pouch young; F1, F5 and F7 (806.7 30.6 g). Saline (sodium chloride injection BP 0.09% 100 ml, David Bull Laboratories, DBL) was used as a control; prostaglandin F2a (Lutalyse 5 mg/ml, UpJohn); and oxytocin (Syntocinon, which contained no vasopressin, 10 IU/ml, Sandoz Pharma). If a result was obtained with both prostaglandin and oxytocin, a prostaglandin synthesis inhibitor (Flunixin meglumine 50 mg/ml Finadyne, Heriot Agvet, Schering, USA) was then administered with oxytocin to determine which of the hormones was responsible for birth-
2.2. Site of injection Individuals were injected intramuscularly into the thigh muscle. Where more than one injection was required to be administered, the injection was alternated between thigh muscles. 2.3. Dosage rates
2.4. Method of recording Preliminary observations of the behavioral components involved with the birthing procedure were noted to enable easy identification of each behavior during the trials. During the experiments, as much behavioral information as possible was recorded including the frequencies, descriptions and durations of behavior. 2.5. Statistical analysis Two-factor ANOVAs were performed for all recorded behaviors with treatment and gender factors. Latency to birth behavior was analysed using the Student's t test. 2.6. Pilot study of other marsupial species A number of marsupial species housed at the University of Tasmania were available for testing (Table 1). A standard dose of PGF2a (0.025 mg kg ÿ 1) or oxytocin (0.5 ± 1 IU) was given intramuscularly in the rump and animals watched for the appearance of birth behavior for the following hour. As this wasapilotstudy,lessbehavioralobservationswereundertaken. 3. Results 3.1. Behavioral descriptions from Per. gunnii Some of the behaviors described here are not solely associated with the birthing procedure but are maintenance
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3.3. Investigative behavior
Table 1 Tasmanian marsupial species used in the pilot study on birth behavior Family
Common name
Scientific name
N
Dasyuridae Dasyuridae Peramelidae Petauridae Phalangeridae Macropodidae Potoroidae Macropodidae
Spotted-tailed quoll Tasmanian devil Southern Brown bandicoot Sugar glider Brushtail possum Tasmanian pademelon Long-nosed potoroo Bennett's wallaby
Dasyurus maculatus Sarcophilus harrissi Isoodon obesulus Petaurus breviceps Tr. vulpecula Thylogale billardierii Potorous tridactylus Macropus rufogriseus
1 4 3 5 4 4 4 2
This is a general behavior and involved investigation of the glass observation arena. Usually, the individual moved around the area sniffing and pawing at the glass, the carpet and the straw on the base. Individuals would sometimes spend large amounts of time in the corners attempting to pull up the carpet. 3.4. Grooming
behaviors. When associated with birth behavior some are performed with increased frequency. These behaviors have not been reported for this species, Per. gunnii, or for any other bandicoot species apart from an actual observation of birth in I. macrourus in captivity [7,11].
This is a normal, maintenance behavior but intensifies and is performed more often when associated with birth. The most common parts of the body that are groomed are, in descending order; the hind, the forepaws, hind flank and face groom. Ear scratching is also often associated with face grooming.
3.2. Sitting and standing This general behavior involves the individual either sitting or standing still for a period of time. Sometimes, the animal may be crouched down as if asleep, at other times, it may be standing on its hind legs with its nose in the air and forefeet poised. Another common position is with the individual on all four feet but one forefoot is raised and the snout is pointed forwards.
3.5. Crouching/prone Crouching involves simply squatting down. However, the prone position involves the animal stretched out with its forefeet out in front and the nose resting on the ground or on the forepaws. This position is an indicator and a forerunner leading to the birth position.
Fig. 1. Photograph of `birth' behavior in a male bandicoot after injection with oxytocin. Note that the scrotum is anterior to the penis in marsupials.
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groomed and licked; in males, the area near the uro-genital sinus and the testes was licked (Fig. 1). During the trials and after injection of prostaglandin or oxytocin, individuals were completely preoccupied in performing the various behaviors and were not distracted by external stimuli. Two-factor ANOVAs for all recorded behaviors gave the following results: There were no significant differences between the sexes in their responses to any of the treatments so their data were pooled. Significant differences in response to treatments did occur for Groom [ F(3,1) = 11.04, P = .0004]; crouching/prone [ F(3,1) = 17.84, P = .0001]; lateral [ F(3,1) = 8.72, P = .0012] and `birth' [ F(3,1) = 3.26, P = .04]. Only the prostaglandin and oxytocin (without Finadyne) treated groups demonstrated the crouching/prone, lateral and `birth' behaviors. Although all groups groomed this was significantly greater in the groups treated with prostaglandin and oxytocin (without Finadyne) (Fig. 2). Fig. 2. Occurrence of behaviors (per 60 min) (mean S.E.) in Per. gunnii (three males and three females) after treatment with saline, prostaglandin, oxytocin and Finadyne followed by oxytocin. Numbers refer to total responding if not all six.
4. The behavioral sequence preceding `Birth' The initial behaviors exhibited when the trial first begins are investigative, sitting and standing. These behaviors appear to last from the beginning until approximately 15 ± 20 min into the trial. Grooming becomes evident at about 13± 14 min and continues for 35 min in males and 20 min in females. There is a slight difference between males and females in relation to the timing of the onset of prone position. Males appear to adopt the prone posture at a later time than females (15 and 10 min, respectively) although this difference is not significant and is possibly the result of the large amount of individual variation encountered throughout the experimental regime. Lateral position occurs at approximately the same time for both sexes (15 min) while birth position and birthing behavior begins at 20 min. All of these behaviors were exhibited in a cyclic fashion and did not occur simultaneously over the whole period.
3.6. Lateral Here, the individual lies on its side, either right lateral or left lateral. This is similar to the normal sleeping posture but differs in that when asleep, they curl up into a tight ball but in the `lateral position' the individual stretches out and does not curl up at all. Grooming of the forepaws, face and hind legs may also be performed while in this position. 3.7. `Birth' The birth position is the same as that adopted for the lateral but the top hind leg is abducted (lifted out of the way) and the area surrounding and leading to the pouch is
Table 2 Pilot study of the effects of PGF2a and oxytocin on Marsupials (includes all previously published data) Family
Common name
Scientific name
N PGF2aa Oxytocina Reference
Macropodidae Potoroidae Macropodidae Potoroidae Macropodidae Phalangeridae Petauridae Peramelidae Peramelidae Dasyuridae Dasyuridae Didelphidaeb
Tammar wallaby Tasmanian bettong Tasmanian pademelon Potoroo Bennett's wallaby Brushtail possum Sugar glider Brown bandicoot Barred bandicoot Tasmanian devil Spotted-tailed quoll Grey opossum
Ma. eugenii B. gaimardi Th. billardierii Po. tridactylus M. rufogriseus Tr. vulpecula Pet. breviceps I. obesulus Per. gunnii S. harrissi D. maculatus Monodelphis domestica
10 15 11 4 4 4 3 5 6 4 1 30
a b c
+ + + + + + + + + + +
ÿ + + + + + + + ÿ +c
[9] [23]; Shaw, 1989; Shaw, personal communication [20] Rose and MacFadyen, unpublished Rose and MacFadyen, unpublished Rose and MacFadyen, unpublished; Loudon and Shaw, unpublished [6]; Rose and MacFadyen, unpublished [20] [6]; Rose and MacFadyen, unpublished This study Rose and MacFadyen, unpublished; This study Rose and MacFadyen, unpublished [21]; Fadem, Rose and Dziadosz, unpublished
Minimal dose of PGF2a that produced an effect was 0.05mg. kg ÿ 1: all oxytocin doses were 1 IU kg ÿ 1. American species, all other are Australian. Males did not respond.
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If these results are compared with the control (saline), it is evident that no birth or components of birth behavior are exhibited (Fig. 2). The responses after an injection of a 0.3 ml dose of oxytocin are displayed in Fig. 2. Not all individuals performed the whole birthing sequence, i.e. birth position; however, components of behaviors including `prone' and `lateral' were performed with regularity. Overall, the adoption of birth position was less frequent for both males and females compared with that exhibited in the PGF2a experiment. The components of birth behavior (crouching/prone, lateral and `birth') did not occur after injection of finadyne and oxytocin. There is no data for the control or Finadyne plus oxytocin treatments for latency (the time interval between injection and the adoption of the birth position) since birth behavior did not occur. Both male and female latencies are similar for the PGF2a trials (18.3 12.3 and 19.3 11.4 min, respectively) but this increased in the oxytocin experiments (32.2 14.2 and 45.4 10.5 min, respectively). There was no significant difference between the sexes for either treatment (PGF2a, P = .29) or (Oxy, P = .39). 4.1. Pilot study All species tested showed clear-cut birth behavior in response to prostaglandin and several did after oxytocin injection (Table 2). 5. Discussion These experiments showed that the barred bandicoot responds to PGF2a by exhibiting birth behavior as previously shown in the tammar wallaby, brush-tail possum and Tasmanian bettong. In fact, every marsupial species that we have tested in our laboratory responds to PGF2a with birth behavior [20]. In addition, we have shown recently that female South American opossums, Mo. domestica also show a birth response to prostaglandin and oxytocin, although males did not respond to oxytocin [21]. We also have shown that birth behavior in the bandicoot can be elicited by oxytocin. Our results suggest that oxytocin acts via the synthesis of PGF2a: We believe this because oxytocin had no effect after prior treatment with Finadyne an inhibitor of PGF2a synthesis. Oxytocin is well known to stimulate the synthesis of PGF2a in many species [4,5]. This would explain the longer latency between injection of oxytocin and the behavioral changes. It was found that the prostaglandin metabolite PGFM was elevated within 5 min after an injection of oxytocin in the red deer Cervus elaphus [5]: In our experiments, the latency increased by approximately 15 ±20 min. The source of the synthesised prostaglandin is unclear; if it is from reproductive tissue, it is surprising that in the Tasmanian bettong, B. gaimardi immature females, pouch young and males respond as quickly as mature females [20]. The role of oxytocin and
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prostaglandins in male marsupials is unclear. However, oxytocin/mesotocin has been found in the testis of the bandicoot I. macrourus [1] and its receptor gene in the prostate of the tammar wallaby [14]. Why male marsupials should respond to prostaglandin and oxytocin with female behavior (birth) is surprising. Perhaps this behavior is `hardwired' in the brain and not easily acted upon by natural selection. Alternatively, the gene(s) responsible may be closely associated with other genes essential for both sexes. The natural levels of prostaglandin and oxytocin might never be sufficiently high to result in the behavior being expressed in males. The role of prostaglandins in the activation of birth behavior in eutherians has been little studied though some eutherian species (e.g. pig, rat and hamster) demonstrate parturient behavior in response to prostaglandin [3,8]: It is also suggested to be an ancient pathway found in some lower vertebrates (fish, amphibia and reptiles) [8]. Oxytocin is known to stimulate maternal behavior in some eutherian mammals [15,16]. It is of great interest that all major groups of Australian marsupials and the one South American form so far tested [21] respond to prostaglandin and oxytocin in a similar way. This suggests that this behavior may have been present in the common ancestor to both groups. One practical application of these results is in the ability to simulate birth in species where a description of such behavior is hitherto lacking. For example, when our experiments were undertaken with Tasmanian devils (S. harrissi) no information on its birth behavior was available. Subsequently, such information has come to light in a film `The Devil's Playground' [12] of a Tasmanian devil giving birth corroborating our results. The finding that prostaglandin precipitates birth behavior [6,20,21,23] has been reproduced in the present study. The finding that oxytocin has a similar effect, only previously shown in the bettong is likely to lead to new areas of research into the relationships between marsupial endocrinology and behavior. For example, the fact that males, females and juvenile marsupials respond in a similar manner suggests that there is an area of the brain in all marsupials that elicits maternal (birth) behavior. The location of this region warrants further study as does the interaction of prostaglandin and oxytocin at birth. Acknowledgments We thank Dr. L. Barmuta for his advice with the statistical analysis and Dr. Sue Jones for her assistance with the experiment. References [1] Bathgate RA, Sernia C, Gemmell RT. Arginine vasopressin- and oxytocin-like peptides in the testis of two Australian marsupials. Peptides 1993;14:701 ± 5.
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[2] Bathgate RA, Parry LJ, Fletcher TP, Shaw G, Renfree MB, Gemmell RT, Sernia C. Comparative aspects of oxytocin-like hormones in marsupials. Adv Exp Med Biol 1995;395:639 ± 55. [3] Blackshaw JK, Blackshaw AW. The effects of prostaglandin PGF2a on the behaviour of the domestic non-pregnant sow and boar. Anim Prod 1982;14:550 ± 2. [4] Flint APF, Sheldrick EL. Ovarian oxytocin and the maternal recognition of pregnancy. J Reprod Fertil 1986;76:831 ± 9. [5] Flint APF, Jabbour HN, Loudon ASI. Oxytocin stimulates uterine prostaglandin F2a in red deer Cervus elaphus. Reprod Fertil Dev 1994;6:269 ± 71. [6] Gemmell RT, MacFadyen AS, Rose RW. The induction of parturient behaviour in possums and bandicoots. Aust Mamm 1991;14:133 ± 6. [7] Gemmell RT, Veith C, Nelson J. Birth in the northern brown bandicoot. Isoodon macrourus (Marsupialia: Peramelidae). Aust J Zool 1999;47:517 ± 28. [8] Guillette LJ, Dubois DH, Cree A. Prostaglandins, oviducal function, and parturient behavior in non-mammalian vertebrates. Am J Physiol 1991;260:R854 ± 61. [9] Hinds LA, Tyndale-Biscoe CH, Shaw G, Fletcher TP, Renfree MB. Effects of prostaglandin and prolactin on luteolysis and parturient behaviour in the non-pregnant tammar, Macropus eugenii. J Reprod Fertil 1990;88:323 ± 33. [10] Lewis PR, Fletcher TP, Renfree MB. Prostaglandin in the peripheral plasma of tammar wallabies at parturition. J Endocrinol 1986;111: 103 ± 9. [11] Lyne AG. Gestation and birth in the marsupial Isoodon macrourus. Aust J Zool 1974;22:303 ± 9. [12] Morris R. The Devil's Playground. Natural History New Zealand Dunedin, NZ. [13] Parry LJ, Guymer F, Fletcher TP, Renfree MB. Release of an oxytocic peptide at parturition in a marsupial, Macropus eugenii. J Reprod Fertil 1996;107:191 ± 8.
[14] Parry LJ, Bathgate RA. Mesotocin receptor gene and protein expression in the prostate gland, but not the testis, of the tammar wallaby, Macropus eugenii. Biol Reprod 1998;59:1101 ± 7. [15] Pederson CA, Prange AJ. Induction of maternal behaviour in virgin female rats after intracerebroventricular administration of oxytocin. Proc Natl Acad Sci U S A 1979;76:6661 ± 5. [16] Pederson CA, Ascher JA, Monroe YL, Prange AJ. Oxytocin induces maternal behaviour in virgin female rats. Science 1982;216:648 ± 50. [17] Renfree MB, Shaw G, Fletcher TP. Evidence for the essential role of prostaglandins for parturition in a marsupial, Macropus eugenii. J Reprod Fertil 1994;102:433 ± 46. [18] Renfree MB, Fletcher TP, Blanden DR, Lewis PR, Shaw G, Gordon K, Short RV, Parer-Cook E, Parer D. Physiological and behavioural events around the time of birth in macropodid marsupials. In: Grigg G, Jarman P, Hume ID, editors. Kangaroos, wallabies and rat ± kangaroos. vol. 1, Sydney, Australia: Surrey Beatty and Sons, 1990; pp. 323 ± 37. [19] Renfree MB, Parry LJ, Fletcher TP, Shaw G. Control of parturition in the marsupial Macropus eugenii. In: Saunders NR, Hinds LA, editors. Recent advances in marsupial biology: University of NSW Press, Sydney, Australia, 1996. pp. 31 ± 40. [20] Rose RW, MacFadyen AS. Oxytocin and prostaglandin F2a induce birth behavior in the bettong Bettongia gaimardi. Horm Behav 1997; 31:120 ± 5. [21] Rose RW, Fadem BH. The hormonal control of birth behavior in the Gray short-tailed opossum (Monodelphis domestica). Horm Behav 2000;37:163 ± 7. [22] Sharman GB, Calaby JH. Reproductive behaviour in the red kangaroo, Megaleia rufa, in captivity. CSIRO Wildl Res 1964;9:58 ± 85. [23] Shaw G. The control of parturient behaviour by prostaglandin F2a in the tammar wallaby (Macropus eugenii). J Reprod Fertil 1990;88: 335 ± 42.