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Mating behaviour of bank voles (Clethrionomys glareolus) modified by hormonal and social factors
Mammalian Biology
Mamm. biol. 68 (2003) 144±152 ã Urban & Fischer Verlag http://www.urbanfischer.de/journals/mammbiol
Zeitschrift fuÈr SaÈugetierkunde
Original investigation
Mating behaviour of bank voles (Clethrionomys glareolus) modified by hormonal and social factors By ANNA MARCHLEWSKA-KOJ, MAèGORZATA KRUCZEK, and P. OLEJNICZAK Institute of Environmental Sciences, Jagiellonian University, KrakoÂw, Poland Receipt of Ms. 25. 03. 2002 Acceptance of Ms. 23. 10. 2002
Abstract The bank vole is a common European rodent. In a natural population the adult females are territorial, and during the breeding season each female's home range overlaps with the home range of several males. The aim of the present study was to investigate the influence of sexual experiences of females and males on mating behaviour. The results indicate that the activity of the female was modified by her sexual experience and by the experience of the partner. Females were more active in presence of sexually non-experienced than experienced males. Males also showed higher activity in the presence of virgin than in the presence of experienced females. Females in postpartum estrus were more active than virgins and injection of gonadotropins stimulated virgin females. Males tested with gonadotropin-treated virgin females or postpartum estrous females showed similar sexual activity. Sexual experience increased the activity of males; they approached receptive females frequently and exhibited a higher number of mountings than did non-experienced males. The higher activity of an experienced partner can prevent copulation with another male during the same estrus, so that only one male becomes the father of the offspring. The present results indicate that in sexually experienced males bulbectomy but not vomeronasalectomy decreased the sexual activity and only 2 out of 6 lesioned males copulated with receptive females. Key words: Clethrionomys glareolus, mating behaviour, sexual experience, olfaction
Introduction The bank vole (Clethrionomys glareolus), a common European rodent, has become a model species in ecological research. Seasonal variation and the influence of social factors on the reproductive activity of the bank vole have been extensively studied in natural populations, and great variation between populations has been observed (for review see: Yoccoz et al. 2000). In a natural population the adult females are territorial, and during the breeding season each female's 1616-5047/03/68/03-144 $ 15.00/0.
home range overlaps with the home range of several males (Bujalska 1973; Bujalska and GruÈm 1989). These voles are relatively easy to breed under laboratory conditions and have become an object of experimentations. Laboratory studies have shown that the hormonal activity of the female is not reflected in vaginal smear fluctuation (Jemioøo et al. 1980) and that the receptivity of the female can be estimated only by her behaviour in the presence of a male
Factors in mating behaviour of voles and by the mounting behaviour of the male. The mating behaviour of bank voles is characterized by a series of multiple brief intromissions and ejaculations (Christiansen and Dùving 1976; Milligan 1979). Ratkiewicz and Borkowska (2000) provided evidence that a female can copulate with and be fertilized by two males during the same receptive period. The present experiments are part of a series of studies on the interaction between bank vole females and males. They were undertaken to investigate the influence of sexual experiences of males on mating behaviour and to determine whether vomeronasalectomy or bulbectomy affect their sexual behaviour.
Material and methods Animals The bank voles used in the experiments came from the Institute of Environmental Sciences, Jagiellonian University in Cracow. They are maintained as outbred stock (heterogeneous but closed population), and we assume that animals from such a colony are comparable to rodents living in a natural population. They were kept in polyethylene cages (36 ´ 21 ´ 17 cm) at 18°±20 °C under 14 h light : 10 h dark (lights on at 6:00 a. m.). Standard pelleted chow and water were available ad libitum. Wood shavings were provided as bedding material. Females and males after weaning at 18±20 days were housed 2±3 per cage in same-sex groups, and 10±12-week-old animals, 19±22 g in body weight, were used for the experiments. Fourteen virgin (Nexp) females were designated for tests with sexually non-experienced (Nexp) or experienced (Exp) males. Nine virgin females were stimulated with gonadotropins; they were injected with 5 iu PMSG (Serogonadotropin, Polfa) 48 h and 5 iu HCG (Chorulon, Polfa) 1 h before behavioural tests. For investigation of receptive females in postpartum estrus, virgin females were paired with males: one female and one male stayed in a cage until pregnancy was apparent. Fourteen pregnant females were separated from the males and kept one animal per cage until parturition. On the day of parturition between 8:00 and 9:00 a.m., the newborns were removed from the mother, and the females in postpartum estrus (Exp) were used for behavioural tests.
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The experiments were carried out on 13 sexually non-experienced (Nexp) males and 29 sexually experienced (Exp) males. The experienced males had copulated with females 18±20 days before testing. To investigate the role of olfactory systems in communication with females during sexual encounters, Exp males were vomeronasalectomized (VNX), bulbectomized (OBX) or shamoperated (SHAM-OBX) and tested in the presence of Exp females. Behavioural tests were carried out two weeks after surgeries.
Behavioural test Males were maintained singly in glass vivariums (40 ´ 20 ´ 25 cm) for 10 days before testing. All behavioural tests were performed between 8:00 a. m. and 3:00 p. m. in the male home cage during 180min. encounters after introduction of a female. Bank voles are nocturnal animals, however, our preliminary observations have shown no differences in their behaviour during the light and dark phases (Marchlewska-Koj unpubl. data). The behaviour of the pair was recorded with a Panasonic WP-CP450/6 video camera and analyzed on a TV monitor. Male sexual activity was measured by the number of mountings of the female during the 180-min. session. The number of female-to-male and male-to-female approaches were also counted during 5-min. samples: 0 to 5 min., 30 to 35 min., 60 to 65 min., 90 to 95 min., 120 to 125 min., and 150 to 155 min. The sum of approaches during the six 5-min. samples was taken as the indicator of total behavioural activity of the female and male during the 180-min. test. Approach was defined as directional locomotion toward the partner to within ca 2 cm. After the termination of behavioural tests vaginal smears were taken from each female and the presence of spermatozoa or a vaginal plug were regarded as evidence of copulation.
Surgical procedures and histological verification All males were anaesthetized with sodium pentobarbitone (0.1 ml/10 g b. w., Polfa, Poland). The vomeronasal organ (VNX) was removed by a standard procedure described for prairie voles (Microtus ochrogaster) by Wysocki et al. (1991). Briefly, the males were placed supine in a stereotaxic apparatus, the mouth was opened and a midline incision was made through the palate. A dental drill was used to remove the exposed bone and the vomeronasal organ was then extracted. The wound was closed with cyanoacrylate gel.
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The bulbectomy (OBX) was performed according to the technique of Leung et al. (1972). Following anaesthesia the males were placed in a stereotaxic apparatus, a cranial midline incision was made, and after the overlying cranial vault was lifted the olfactory bulbs were removed by aspiration. The residual cavity was filled with gel-foam. The anaesthesia and incision of the sham surgical procedure matched the OBX protocol; the olfactory bulbs remained intact. For histological verification of VNX and OBX the animals were deeply anaesthetized with sodium pentobarbitone and perfused through the heart with physiological saline followed by fixative (4% formaldehyde). The brains of the VNX males were removed and embedded in paraffin wax. The olfactory bulbs and frontal poles of the brains were serially sectioned in the horizontal plane at 10 lm and stained with Delafied's haematoxylin and eosin. The removal of the vomeronasal organ was verified by the complete absence of glomeruli in the accessory olfactory bulbs. In this study only one male (out of six) had incomplete VNX and behavioural data collected from this animal was not considered. The brains of the OBX males were carefully removed from the skulls and the extent of bulb removal was microscopically checked. In the present experiments all bulbectomized males obtained complete olfactory bulb removal. The experimental procedures for the present study were approved by the Jagiellonian University Rector's Permanent Committee on the Bioethics of Animal Experimentation.
Statistical analysis Behavioural data were analyzed by two-way analysis of variance and one-way analysis of variance, and post hoc comparisons were made with Tukey's test for unequal sample sizes (Statistica, v. 5.5)
Results The number of approaches during the first 5 min. and the sum of approaches during the six 5-min. samples were taken as indicator of total behavioural activity of the female and male during the 180-min. encounters. The results summarized in table 1 and analyzed by two-way ANOVA indicate that the activity of the female was modified by her sexual experience and by the experience of the presented partner during the first 5 min. (df 3.24; F = 22.49, p < 0.0001 and df 3, 24; F = 10.99751, p < 0.01, respectively) and during the whole encounter (df 3.24; F = 13.10049, p < 0.001, and df 3.24; F = 11.2328, p < 0.005). Sexually experienced males also showed higher activity in the presence of virgin than in the presence of experienced females (df 3.24; F = 9.4026, p < 0.01) during the first 5 min. of the test. The results summarized in table 2 indicate that only 2 out of 7 non-experienced pairs (Nexp female and Nexp male) mated, whereas most of the sexually non-experienced or experienced males copulated when paired with sexually experienced females, and experienced males showed shorter latency to the first mounting and a higher number of mountings than did nonexperienced males. All females that had copulated were fertilized and gave birth 18 to 20 days after the behavioural test. The behavioural activity of females in the presence of sexual experienced males is summarized in figure 1. Females in postpartum estrus were more active than virgins during the first 5 min. of encounters (df
Table 1. Behavioural activity estimated by number of approaches of bank vole females and males during 180min. encounters. Virgin non-experienced (Nexp) females or experienced postpartum estrous (Exp) females were paired with non-experienced (Nexp) or experienced (Exp) males. (Means ± SE) Means marked by the same symbols differ significantly from each other Females
Males
No. of pairs
Activity of females
Activity of males
First 5 min.
Total
First 5 min.
Total
Nexp Nexp Exp Exp
Nexp Exp Nexp Exp
7 7 6 8
11.6 ± 3.8a 2.9 ± 0.9a 33.0 ± 7.6b 11.9 ± 1.4b
55.0 ± 10.8a 18.7 ± 5.5a 80.3 ± 10.5b 53.6 ± 8.3b
43.7 ± 14.8 43.6 ± 13.7 4.2 ± 1.9a 27.4 ± 9.9a
182.4 ± 59.0 218.1 ± 67.0 93.7 ± 43.4 150.4 ± 28.1
Factors in mating behaviour of voles 2.18; F = 3.9842, p < 0.05). Injection of gonadotropins stimulated virgin females. This was manifested in an increased number of approaches toward males (Fig. 1 A), but the difference was not significant. Gonado-
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tropins stimulated the receptivity of virgin females, and 8 out of 9 tested females copulated (Tab. 3). All females delivered pups on the 19th day after behavioural tests. Males tested with gonadotropin-treated vir-
Table 2. Sexual behaviour of males estimated by latency and number of mountings during 180-min. encounters. Non-experienced (Nexp) or experienced (Exp) males were paired with virgin non-experienced (Nexp) females or experienced postpartum estrous (Exp) females Females
Males
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Nexp
Nexp
7
2
42 (4. 80)
153.5 (67. 240)
Nexp
Exp
7
4
25.2 (1±51)
21.2 (4±60)
Exp
Nexp
6
4
47.0 (14±80)
32.0 (2±72)
Exp
Exp
8
7
9.1 (1±15)
73.0 (26±121)
Fig. 1. Behavioural activity of females estimated by number of approaches to sexually experienced males during first 5 min. (A) and 180-min. encounters (B). Virgin non-experienced (Nexp) females, experienced postpartum estrous (Exp) females, or virgin, non-experienced females injected with gonadotropin (PMSG + HCG) were tested. Means ± SE.
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gin females or postpartum estrous females showed similar sexual activity, as measured by the duration of latency to the first mounting and the number of mountings during the 180 min. session. Bulbectomy (OBX) but not vomeronasalectomy (VNX) affected the behaviour of
males in the presence of receptive, postpartum estrous, females (Fig. 2). OBX males showed lower activity than VNX or shamoperated (SHAM) males during the first 5 min. (df 2.16; F = 23.5000, p < 0.0001, Tukey's test p < 0.01) (Fig. 2 A) and total activity during the tests (df 2.16; F = 13.1607,
Table 3. Sexual behaviour of sexually experienced (Exp) males estimated by latency and number of mountings of females during 180-min. encounters. Virgin non-experienced (Nexp) females, experienced postpartum estrous (Exp) females, or virgin non-experienced females injected with gonadotropins (PMSG + HCG) were tested Males
Females
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Exp
Nexp
7
4
25.2 (1±51)
21.2 (4±60)
Exp
Exp
5
5
4.2 (2±9)
87.0 (20±112)
Exp
PMSG + HCG
9
8
2.2 (1±5)
73.7 (7±211)
Fig. 2. Behavioural activity of experienced males sham-operated (Sham), vomeronasalectomized (VNX), or bulbectomized (OBX), paired with sexually experienced postpartum estrous females, estimated by number of approaches during first 5min. (A) and 180-min. encounters (B). Means ± SE.
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Table 4. Copulatory behaviour of males estimated by latency and number of mountings during 180-min. encounters. Subjects were experienced males: sham-operated (Sham), vomeronasalectomized (VNX) or bulbectomized (OBX). They were paired with experienced postpartum estrous females during 180-min. encounters Males
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Sham
8
8
4.4 (1±8)
65 (26±78)
VNX
5
4
8.5 (3±24)
82.7 (43±114)
OBX
6
2
29 (10. 48)
11.5 (3. 20)
p < 0.0005, Tukey's test p < 0.05) (Fig. 2 B). All SHAM and 4 out of 5 tested VNX males copulated with receptive, postpartum estrous, females. The latency to the first mounting and the number of mountings were similar between these experimental groups (Tab. 4). Bulbectomy did not impair copulatory behaviour in 2 out of 6 OBX tested males. The number of mountings observed for OBX males was lower than for SHAM or VNX males, but all females that had copulated were fertilized and delivered pups at 18 to 20 days after copulation. All males designed for bulbectomy and 70% for vomeronasalectomy survived the surgeries.
Discussion Clethrionomys glareolus females, similarly to other Microtidae, are characterized by provoked ovulation: ovulation is induced by mating and occurs 6 to 14 hours after coitus (Clarke et al. 1970; Clarke 1985). However, under laboratory conditions a female kept in the presence of a male (located in a small wire-mesh cage) showed spontaneous ovulation during the next 4 to 6 days after introduction of the male. Copulation did not take place, but tactile, olfactory, visual and auditory stimuli were exchanged between them (Clarke and Hellwing 1977). A similar male effect was observed in female field voles (Microtus
agrestis), another induced-ovulating species (Milligan 1974). In bank vole females, reproductive activity is stimulated by male pheromones and appears as an increased number of Graafian follicles in adult females exposed to the male or male urine, but ovulation does not occur (Jemioøo et al. 1980; Marchlewska-Koj 2001). Ovulation can be evoked in adult bank vole females by injection of FSH and LH gonadotropins. In 7 out of 12 females, corpora lutea (3.5 ± 0.1 corpora lutea per female) with healthy luteal cells and former follicular antria were found 14 hours after LH treatment. In ovaries of 10 females injected with physiological saline, fresh corpora lutea were not identified (Marchlewska-Koj unpubl. data). The present results indicate that injection of gonadotropins stimulated female receptivity during the next 3 hours after HCG treatment. Moreover, bank vole females, like other rodents, show postpartum estrus appearing as receptivity during a few hours after parturition. The present data confirmed that the hormonal activity of females modified their behaviour and that receptive females more frequently approached sexual partners than did virgin females. Sexual experience increased the activity of males; they approached receptive females frequently and mounted after a short latency. Experienced males also exhibited a higher number of mountings than did nonexperienced males, when they were paired
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with receptive females, however highest mounting frequency was demonstrated by non-experienced males paired with virgin females. Mountings of experienced males were recorded during 30 to 90 min. of tests (data not shown). Our results are consistent with the observations of Ratkiewicz and Borkowska (2000) and indicate that females are receptive for a relatively long period. The higher activity of an experienced partner may prevent copulation with another male during the same estrus, so that only one male is more likely to become the father of all offspring in the litter. There is no doubt that male olfactory signals affect the behaviour of females, and bank vole females are more active in the presence of dominant than in the presence of subordinate or castrated males (Hoffmeyer 1982; Kruczek and Pochron 1997). The behaviour of receptive females and their pheromones presumably are signals which stimulate the behaviour of males. Males discriminated the sex of a partner by pheromones and exhibited aggression only toward other adult males, but their behaviour was changed after bulbectomy (Marchlewska-Koj et al. 1989; Kapusta et al. 1996). Bank vole males emitted ultrasounds at frequencies of 30 to 45 KHz, responding to the presence of females or bedding of adult females. This stimulating effect of female pheromones was noticed in non-operated or vomeronasalectomized males, but ultrasounds were not recorded in bulbectomized animals (Kapusta et al. 1996). The present data confirm previous results indicating that bulbectomy but not vomeronasalectomy decrease the behav-
ioural activity of males in the presence of females. An OBX male paired with a receptive female showed significantly fewer approaches to the partner; only 2 out of 6 lesioned males copulated, and made only a few mountings. These observations are consistent with the behavioural effects of bulbectomy in Microtus ochrogaster (Kirkpatrick et al. 1994). Lesion of olfactory systems decreased but did not always inhibit copulation of males; this indicates that copulation can take place without olfactory contact. Complete bulbectomy is associated with the removal of the main and the vomeronasal olfactory systems. The behavioural effect of bulbectomy may result from damage to any of these systems. Our results show that the behaviour of vomeronasalectomized males paired with a receptive females was similar to that of SHAMoperated animals. It can be suggested that missing input from the vomeronasal organ can be compensated by the main olfactory epithelium. Moreover, there is some evidence that the functions of the main and vomeronasal olfactory systems are sometimes overlapping and sometimes discrete. Male golden hamster copulatory behaviour was initially thought to be mediated primarily by the vomeronasal system, but now it is clear that both systems are important (Meredith 1980, 1983).
Acknowledgements This work was supported by a grant from the State Committee for Scientific Research, Poland, KBN PB 0919/P04/98/15.
Zusammenfassung Paarungsverhalten der RoÈtelmaus (Clethrionomys glareolus) unter Einfluû hormonaler und sozialer Faktoren Die RoÈtelmaus ist ein weit verbreitetes europaÈisches Nagetier. In natuÈrlichen Populationen zeigen erwachsene Weibchen TerritorialitaÈt, und waÈhrend der Brunstzeit uÈberlappt das Revier jeden Weibchens das mehrerer MaÈnnchen. Ziel dieser Studie war die Untersuchung des Einflusses der sexuellen Erfahrung von Weibchen und MaÈnnchen auf das Paarungsverhalten. Die Ergebnisse zeigen, daû die AktivitaÈt der Weibchen durch deren sexuelle Erfahrung und durch die des Partners modifiziert war.
Factors in mating behaviour of voles
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Die MaÈnnchen zeigten auch groÈûere AktivitaÈt in der Gegenwart des einen virginellen Weibchens als in der von erfahrenen Weibchen. Weibchen in post-partum Ústrus waren aktiver als virginelle Weibchen, und Gonadotropin-Injektionen wirkten auf die virginellen Weibchen stimulierend. MaÈnnchen zeigten in Anwesenheit von jungfraÈulichen Weibchen, die mit Gonadotropin-Injektionen behandelt waren und gegenuÈber post-partum-Ústrus-Weibchen aÈhnliche sexuelle AktivitaÈt. Sexuelle Erfahrung verstaÈrkte die AktivitaÈt der MaÈnnchen. Sie naÈherten sich haÈufig den rezeptiven Weibchen und zeigten mehr Kopulationsversuche als unerfahrene MaÈnnchen. HoÈhere AktivitaÈt eines erfahrenen Partners kann moÈglicherweise die Kopulation mit einem anderen MaÈnnchen waÈhrend ein und desselben Ústrus verhindern, wodurch der Nachwuchs nur von einem MaÈnnchen erzeugt wird. Die vorliegenden Ergebnisse zeigen, daû Bulbektomie, nicht aber Vomeronasalektomie bei sexuell erfahrenen MaÈnnchen die sexuelle AktivitaÈt verminderte. Nur 2 von 6 der LaÈsion unterzogenen MaÈnnchen kopulierten mit rezeptiven Weibchen.
References Bujalska, G. (1973): The role of spacing behaviour among female in the regulation of the reproduction in the bank vole. J. Reprod. Fert. suppl. 19, 463±472. Bujalska, G.; GruÈm, L. (1989): Social organization of the bank vole (Clethrionomys glareolus Schreber, 1780) and its demographic consequences: a model. Oecologia (Berlin) 80, 70±81. Christiansen, E.; Dùving, K. B. (1976): Observations of the mating behaviour of the bank vole, Clethrionomys glareolus. Behav. Biol. 17, 263±266. Clarke, J. R. (1985): The reproductive biology of the bank vole (Clethrionomys glareolus) and the wood mouse (Apodemus sylvaticus). Symp. zool. Soc. London 55, 33±59. Clarke, J. R.; Clulow, F. V.; Greig, F. (1970): Ovulation in the bank vole, Clethrionomys glareolus. J. Reprod. Fert. 23, 531. Clarke, J. R.; Hellwing, S. (1977): Remote control by males of ovulation in bank voles (Clethrionomys glareolus). J. Reprod. Fert. 50, 155±158. Hoffmeyer, I. (1982): Responses of female bank voles (Clethrionomys glareolus) to dominant vs subordinate conspecific males. Behav. Neural. Biol. 36, 178±188. Jemioøo, B.; Marchlewska-Koj, A.; Buchalczyk, A. (1980): Acceleration of ovarian follicle maturation of female caused by male in Microtus agrestis and Clethrionomys glareolus. Folia Biol. (Krakow) 28, 269±277. Kapusta, J.; Marchlewska-Koj, A.; Olejniczak, P.; Kruczek, M. (1996): Removal of the olfactory system modifies male bank vole behaviour in presence of females. Behav. Process. 37, 39±45.
Kirkpatrick, B.; Williams, J. R.; Slotnick, B. M.; Carter, C. S. (1994): Olfactory bulbectomy decreases social behavior in male prairie voles (M. ochrogaster). Physiol. Behav. 55, 885±889. Kruczek, M.; PochronÂ, E. (1997): Chemical signals from conspecifics modify the activity of female bank voles, Clethrionomys glareolus. Acta Theriol. 42, 71±78. Leung, P. M.; Larson, D. M.; Rogers, Q. R. (1972): Food intake and preference of olfactory bulbectomized rats fed amino acid imbalanced or deficient diets. Physiol. Behav. 15, 381±387. Marchlewska-Koj, A.; Koøodziej, B.; Filimowska, A. (1989): Aggressive behavior of adult bank voles (Clethrionomys glareolus) towards conspecifics. Aggress. Behav. 15, 381±387. Marchlewska-Koj, A. (2001): Pheromones regulation of bank vole (Clethrionomys glareolus) reproduction. In: Chemical Signals in Vertebrates. Vol. 9. Ed. by A. Marchlewska-Koj, J. J. Lepri and D. MuÈller-Schwarze. New York: Kluwer/Plenum Publishers. Pp. 391±396. Meredith, M. (1980): The vomeronasal organ and accessory olfactory system in the hamster. In: Chemical Signals in Vertebrates and Aquatic Invertebrates. Ed. by D. MuÈller-Schwarze and R. Silverstein. New York: Plenum Press. Pp. 303±326. Meredith, M. (1983): Sensory physiology of pheromone communication. In: Pheromones and Reproduction in Mammals. Ed. by J. G. Vandenbergh. New York: Academic Press. Pp. 200± 252. Milligan, R. S. (1974): Social environment and ovulation in the vole, Microtus agrestis. J. Reprod. Fert. 41, 35±47. Milligan, R. S. (1979): The copulatory pattern of the bank vole (Clethrionomys glareolus) and
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speculation on the role of penile spines. J. Zool. (London) 188, 279±300. Ratkiewicz, M.; Borkowska, A. (2000): Multiple paternity in bank vole (Cletrionomys glareolus); field and experimental data. Z. SaÈugetierkunde 65, 6±14. Wysocki, C. J.; Kruczek, M.; Wysocki, L.; Lepri, J. J. (1991): Activation of reproduction in nulliparous and primiparous voles is blocked by vomeronasal organ removal. Biol. Reprod. 45, 611±616. Yoccoz, N. G.; Hansson, L.; Ims, R. A. (2000): Geographical differences in size, reproduction and behaviour of bank voles in relation to density variations. Pol. J. Ecol. 48 suppl., 63±74.
Authors' addresses: Anna Marchlewska-Koj, Maøgorzata Kruczek, Institute of Environmental Sciences, Jagiellonian University, ul. Ingardena 6, 30-060 KrakoÂw, Poland (e-mail: [email protected]); Paweø Olejniczak, Institute of Nature Conservation, Polish Academy of Sciences, Mickiewicza 33, 31-120 KrakoÂw, Poland
Mamm. biol. 68 (2003) 144±152 ã Urban & Fischer Verlag http://www.urbanfischer.de/journals/mammbiol
Zeitschrift fuÈr SaÈugetierkunde
Original investigation
Mating behaviour of bank voles (Clethrionomys glareolus) modified by hormonal and social factors By ANNA MARCHLEWSKA-KOJ, MAèGORZATA KRUCZEK, and P. OLEJNICZAK Institute of Environmental Sciences, Jagiellonian University, KrakoÂw, Poland Receipt of Ms. 25. 03. 2002 Acceptance of Ms. 23. 10. 2002
Abstract The bank vole is a common European rodent. In a natural population the adult females are territorial, and during the breeding season each female's home range overlaps with the home range of several males. The aim of the present study was to investigate the influence of sexual experiences of females and males on mating behaviour. The results indicate that the activity of the female was modified by her sexual experience and by the experience of the partner. Females were more active in presence of sexually non-experienced than experienced males. Males also showed higher activity in the presence of virgin than in the presence of experienced females. Females in postpartum estrus were more active than virgins and injection of gonadotropins stimulated virgin females. Males tested with gonadotropin-treated virgin females or postpartum estrous females showed similar sexual activity. Sexual experience increased the activity of males; they approached receptive females frequently and exhibited a higher number of mountings than did non-experienced males. The higher activity of an experienced partner can prevent copulation with another male during the same estrus, so that only one male becomes the father of the offspring. The present results indicate that in sexually experienced males bulbectomy but not vomeronasalectomy decreased the sexual activity and only 2 out of 6 lesioned males copulated with receptive females. Key words: Clethrionomys glareolus, mating behaviour, sexual experience, olfaction
Introduction The bank vole (Clethrionomys glareolus), a common European rodent, has become a model species in ecological research. Seasonal variation and the influence of social factors on the reproductive activity of the bank vole have been extensively studied in natural populations, and great variation between populations has been observed (for review see: Yoccoz et al. 2000). In a natural population the adult females are territorial, and during the breeding season each female's 1616-5047/03/68/03-144 $ 15.00/0.
home range overlaps with the home range of several males (Bujalska 1973; Bujalska and GruÈm 1989). These voles are relatively easy to breed under laboratory conditions and have become an object of experimentations. Laboratory studies have shown that the hormonal activity of the female is not reflected in vaginal smear fluctuation (Jemioøo et al. 1980) and that the receptivity of the female can be estimated only by her behaviour in the presence of a male
Factors in mating behaviour of voles and by the mounting behaviour of the male. The mating behaviour of bank voles is characterized by a series of multiple brief intromissions and ejaculations (Christiansen and Dùving 1976; Milligan 1979). Ratkiewicz and Borkowska (2000) provided evidence that a female can copulate with and be fertilized by two males during the same receptive period. The present experiments are part of a series of studies on the interaction between bank vole females and males. They were undertaken to investigate the influence of sexual experiences of males on mating behaviour and to determine whether vomeronasalectomy or bulbectomy affect their sexual behaviour.
Material and methods Animals The bank voles used in the experiments came from the Institute of Environmental Sciences, Jagiellonian University in Cracow. They are maintained as outbred stock (heterogeneous but closed population), and we assume that animals from such a colony are comparable to rodents living in a natural population. They were kept in polyethylene cages (36 ´ 21 ´ 17 cm) at 18°±20 °C under 14 h light : 10 h dark (lights on at 6:00 a. m.). Standard pelleted chow and water were available ad libitum. Wood shavings were provided as bedding material. Females and males after weaning at 18±20 days were housed 2±3 per cage in same-sex groups, and 10±12-week-old animals, 19±22 g in body weight, were used for the experiments. Fourteen virgin (Nexp) females were designated for tests with sexually non-experienced (Nexp) or experienced (Exp) males. Nine virgin females were stimulated with gonadotropins; they were injected with 5 iu PMSG (Serogonadotropin, Polfa) 48 h and 5 iu HCG (Chorulon, Polfa) 1 h before behavioural tests. For investigation of receptive females in postpartum estrus, virgin females were paired with males: one female and one male stayed in a cage until pregnancy was apparent. Fourteen pregnant females were separated from the males and kept one animal per cage until parturition. On the day of parturition between 8:00 and 9:00 a.m., the newborns were removed from the mother, and the females in postpartum estrus (Exp) were used for behavioural tests.
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The experiments were carried out on 13 sexually non-experienced (Nexp) males and 29 sexually experienced (Exp) males. The experienced males had copulated with females 18±20 days before testing. To investigate the role of olfactory systems in communication with females during sexual encounters, Exp males were vomeronasalectomized (VNX), bulbectomized (OBX) or shamoperated (SHAM-OBX) and tested in the presence of Exp females. Behavioural tests were carried out two weeks after surgeries.
Behavioural test Males were maintained singly in glass vivariums (40 ´ 20 ´ 25 cm) for 10 days before testing. All behavioural tests were performed between 8:00 a. m. and 3:00 p. m. in the male home cage during 180min. encounters after introduction of a female. Bank voles are nocturnal animals, however, our preliminary observations have shown no differences in their behaviour during the light and dark phases (Marchlewska-Koj unpubl. data). The behaviour of the pair was recorded with a Panasonic WP-CP450/6 video camera and analyzed on a TV monitor. Male sexual activity was measured by the number of mountings of the female during the 180-min. session. The number of female-to-male and male-to-female approaches were also counted during 5-min. samples: 0 to 5 min., 30 to 35 min., 60 to 65 min., 90 to 95 min., 120 to 125 min., and 150 to 155 min. The sum of approaches during the six 5-min. samples was taken as the indicator of total behavioural activity of the female and male during the 180-min. test. Approach was defined as directional locomotion toward the partner to within ca 2 cm. After the termination of behavioural tests vaginal smears were taken from each female and the presence of spermatozoa or a vaginal plug were regarded as evidence of copulation.
Surgical procedures and histological verification All males were anaesthetized with sodium pentobarbitone (0.1 ml/10 g b. w., Polfa, Poland). The vomeronasal organ (VNX) was removed by a standard procedure described for prairie voles (Microtus ochrogaster) by Wysocki et al. (1991). Briefly, the males were placed supine in a stereotaxic apparatus, the mouth was opened and a midline incision was made through the palate. A dental drill was used to remove the exposed bone and the vomeronasal organ was then extracted. The wound was closed with cyanoacrylate gel.
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The bulbectomy (OBX) was performed according to the technique of Leung et al. (1972). Following anaesthesia the males were placed in a stereotaxic apparatus, a cranial midline incision was made, and after the overlying cranial vault was lifted the olfactory bulbs were removed by aspiration. The residual cavity was filled with gel-foam. The anaesthesia and incision of the sham surgical procedure matched the OBX protocol; the olfactory bulbs remained intact. For histological verification of VNX and OBX the animals were deeply anaesthetized with sodium pentobarbitone and perfused through the heart with physiological saline followed by fixative (4% formaldehyde). The brains of the VNX males were removed and embedded in paraffin wax. The olfactory bulbs and frontal poles of the brains were serially sectioned in the horizontal plane at 10 lm and stained with Delafied's haematoxylin and eosin. The removal of the vomeronasal organ was verified by the complete absence of glomeruli in the accessory olfactory bulbs. In this study only one male (out of six) had incomplete VNX and behavioural data collected from this animal was not considered. The brains of the OBX males were carefully removed from the skulls and the extent of bulb removal was microscopically checked. In the present experiments all bulbectomized males obtained complete olfactory bulb removal. The experimental procedures for the present study were approved by the Jagiellonian University Rector's Permanent Committee on the Bioethics of Animal Experimentation.
Statistical analysis Behavioural data were analyzed by two-way analysis of variance and one-way analysis of variance, and post hoc comparisons were made with Tukey's test for unequal sample sizes (Statistica, v. 5.5)
Results The number of approaches during the first 5 min. and the sum of approaches during the six 5-min. samples were taken as indicator of total behavioural activity of the female and male during the 180-min. encounters. The results summarized in table 1 and analyzed by two-way ANOVA indicate that the activity of the female was modified by her sexual experience and by the experience of the presented partner during the first 5 min. (df 3.24; F = 22.49, p < 0.0001 and df 3, 24; F = 10.99751, p < 0.01, respectively) and during the whole encounter (df 3.24; F = 13.10049, p < 0.001, and df 3.24; F = 11.2328, p < 0.005). Sexually experienced males also showed higher activity in the presence of virgin than in the presence of experienced females (df 3.24; F = 9.4026, p < 0.01) during the first 5 min. of the test. The results summarized in table 2 indicate that only 2 out of 7 non-experienced pairs (Nexp female and Nexp male) mated, whereas most of the sexually non-experienced or experienced males copulated when paired with sexually experienced females, and experienced males showed shorter latency to the first mounting and a higher number of mountings than did nonexperienced males. All females that had copulated were fertilized and gave birth 18 to 20 days after the behavioural test. The behavioural activity of females in the presence of sexual experienced males is summarized in figure 1. Females in postpartum estrus were more active than virgins during the first 5 min. of encounters (df
Table 1. Behavioural activity estimated by number of approaches of bank vole females and males during 180min. encounters. Virgin non-experienced (Nexp) females or experienced postpartum estrous (Exp) females were paired with non-experienced (Nexp) or experienced (Exp) males. (Means ± SE) Means marked by the same symbols differ significantly from each other Females
Males
No. of pairs
Activity of females
Activity of males
First 5 min.
Total
First 5 min.
Total
Nexp Nexp Exp Exp
Nexp Exp Nexp Exp
7 7 6 8
11.6 ± 3.8a 2.9 ± 0.9a 33.0 ± 7.6b 11.9 ± 1.4b
55.0 ± 10.8a 18.7 ± 5.5a 80.3 ± 10.5b 53.6 ± 8.3b
43.7 ± 14.8 43.6 ± 13.7 4.2 ± 1.9a 27.4 ± 9.9a
182.4 ± 59.0 218.1 ± 67.0 93.7 ± 43.4 150.4 ± 28.1
Factors in mating behaviour of voles 2.18; F = 3.9842, p < 0.05). Injection of gonadotropins stimulated virgin females. This was manifested in an increased number of approaches toward males (Fig. 1 A), but the difference was not significant. Gonado-
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tropins stimulated the receptivity of virgin females, and 8 out of 9 tested females copulated (Tab. 3). All females delivered pups on the 19th day after behavioural tests. Males tested with gonadotropin-treated vir-
Table 2. Sexual behaviour of males estimated by latency and number of mountings during 180-min. encounters. Non-experienced (Nexp) or experienced (Exp) males were paired with virgin non-experienced (Nexp) females or experienced postpartum estrous (Exp) females Females
Males
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Nexp
Nexp
7
2
42 (4. 80)
153.5 (67. 240)
Nexp
Exp
7
4
25.2 (1±51)
21.2 (4±60)
Exp
Nexp
6
4
47.0 (14±80)
32.0 (2±72)
Exp
Exp
8
7
9.1 (1±15)
73.0 (26±121)
Fig. 1. Behavioural activity of females estimated by number of approaches to sexually experienced males during first 5 min. (A) and 180-min. encounters (B). Virgin non-experienced (Nexp) females, experienced postpartum estrous (Exp) females, or virgin, non-experienced females injected with gonadotropin (PMSG + HCG) were tested. Means ± SE.
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gin females or postpartum estrous females showed similar sexual activity, as measured by the duration of latency to the first mounting and the number of mountings during the 180 min. session. Bulbectomy (OBX) but not vomeronasalectomy (VNX) affected the behaviour of
males in the presence of receptive, postpartum estrous, females (Fig. 2). OBX males showed lower activity than VNX or shamoperated (SHAM) males during the first 5 min. (df 2.16; F = 23.5000, p < 0.0001, Tukey's test p < 0.01) (Fig. 2 A) and total activity during the tests (df 2.16; F = 13.1607,
Table 3. Sexual behaviour of sexually experienced (Exp) males estimated by latency and number of mountings of females during 180-min. encounters. Virgin non-experienced (Nexp) females, experienced postpartum estrous (Exp) females, or virgin non-experienced females injected with gonadotropins (PMSG + HCG) were tested Males
Females
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Exp
Nexp
7
4
25.2 (1±51)
21.2 (4±60)
Exp
Exp
5
5
4.2 (2±9)
87.0 (20±112)
Exp
PMSG + HCG
9
8
2.2 (1±5)
73.7 (7±211)
Fig. 2. Behavioural activity of experienced males sham-operated (Sham), vomeronasalectomized (VNX), or bulbectomized (OBX), paired with sexually experienced postpartum estrous females, estimated by number of approaches during first 5min. (A) and 180-min. encounters (B). Means ± SE.
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Table 4. Copulatory behaviour of males estimated by latency and number of mountings during 180-min. encounters. Subjects were experienced males: sham-operated (Sham), vomeronasalectomized (VNX) or bulbectomized (OBX). They were paired with experienced postpartum estrous females during 180-min. encounters Males
No. of males
Mountings
Tested
Copulated
Latency (sec) (range)
Number (range)
Sham
8
8
4.4 (1±8)
65 (26±78)
VNX
5
4
8.5 (3±24)
82.7 (43±114)
OBX
6
2
29 (10. 48)
11.5 (3. 20)
p < 0.0005, Tukey's test p < 0.05) (Fig. 2 B). All SHAM and 4 out of 5 tested VNX males copulated with receptive, postpartum estrous, females. The latency to the first mounting and the number of mountings were similar between these experimental groups (Tab. 4). Bulbectomy did not impair copulatory behaviour in 2 out of 6 OBX tested males. The number of mountings observed for OBX males was lower than for SHAM or VNX males, but all females that had copulated were fertilized and delivered pups at 18 to 20 days after copulation. All males designed for bulbectomy and 70% for vomeronasalectomy survived the surgeries.
Discussion Clethrionomys glareolus females, similarly to other Microtidae, are characterized by provoked ovulation: ovulation is induced by mating and occurs 6 to 14 hours after coitus (Clarke et al. 1970; Clarke 1985). However, under laboratory conditions a female kept in the presence of a male (located in a small wire-mesh cage) showed spontaneous ovulation during the next 4 to 6 days after introduction of the male. Copulation did not take place, but tactile, olfactory, visual and auditory stimuli were exchanged between them (Clarke and Hellwing 1977). A similar male effect was observed in female field voles (Microtus
agrestis), another induced-ovulating species (Milligan 1974). In bank vole females, reproductive activity is stimulated by male pheromones and appears as an increased number of Graafian follicles in adult females exposed to the male or male urine, but ovulation does not occur (Jemioøo et al. 1980; Marchlewska-Koj 2001). Ovulation can be evoked in adult bank vole females by injection of FSH and LH gonadotropins. In 7 out of 12 females, corpora lutea (3.5 ± 0.1 corpora lutea per female) with healthy luteal cells and former follicular antria were found 14 hours after LH treatment. In ovaries of 10 females injected with physiological saline, fresh corpora lutea were not identified (Marchlewska-Koj unpubl. data). The present results indicate that injection of gonadotropins stimulated female receptivity during the next 3 hours after HCG treatment. Moreover, bank vole females, like other rodents, show postpartum estrus appearing as receptivity during a few hours after parturition. The present data confirmed that the hormonal activity of females modified their behaviour and that receptive females more frequently approached sexual partners than did virgin females. Sexual experience increased the activity of males; they approached receptive females frequently and mounted after a short latency. Experienced males also exhibited a higher number of mountings than did nonexperienced males, when they were paired
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with receptive females, however highest mounting frequency was demonstrated by non-experienced males paired with virgin females. Mountings of experienced males were recorded during 30 to 90 min. of tests (data not shown). Our results are consistent with the observations of Ratkiewicz and Borkowska (2000) and indicate that females are receptive for a relatively long period. The higher activity of an experienced partner may prevent copulation with another male during the same estrus, so that only one male is more likely to become the father of all offspring in the litter. There is no doubt that male olfactory signals affect the behaviour of females, and bank vole females are more active in the presence of dominant than in the presence of subordinate or castrated males (Hoffmeyer 1982; Kruczek and Pochron 1997). The behaviour of receptive females and their pheromones presumably are signals which stimulate the behaviour of males. Males discriminated the sex of a partner by pheromones and exhibited aggression only toward other adult males, but their behaviour was changed after bulbectomy (Marchlewska-Koj et al. 1989; Kapusta et al. 1996). Bank vole males emitted ultrasounds at frequencies of 30 to 45 KHz, responding to the presence of females or bedding of adult females. This stimulating effect of female pheromones was noticed in non-operated or vomeronasalectomized males, but ultrasounds were not recorded in bulbectomized animals (Kapusta et al. 1996). The present data confirm previous results indicating that bulbectomy but not vomeronasalectomy decrease the behav-
ioural activity of males in the presence of females. An OBX male paired with a receptive female showed significantly fewer approaches to the partner; only 2 out of 6 lesioned males copulated, and made only a few mountings. These observations are consistent with the behavioural effects of bulbectomy in Microtus ochrogaster (Kirkpatrick et al. 1994). Lesion of olfactory systems decreased but did not always inhibit copulation of males; this indicates that copulation can take place without olfactory contact. Complete bulbectomy is associated with the removal of the main and the vomeronasal olfactory systems. The behavioural effect of bulbectomy may result from damage to any of these systems. Our results show that the behaviour of vomeronasalectomized males paired with a receptive females was similar to that of SHAMoperated animals. It can be suggested that missing input from the vomeronasal organ can be compensated by the main olfactory epithelium. Moreover, there is some evidence that the functions of the main and vomeronasal olfactory systems are sometimes overlapping and sometimes discrete. Male golden hamster copulatory behaviour was initially thought to be mediated primarily by the vomeronasal system, but now it is clear that both systems are important (Meredith 1980, 1983).
Acknowledgements This work was supported by a grant from the State Committee for Scientific Research, Poland, KBN PB 0919/P04/98/15.
Zusammenfassung Paarungsverhalten der RoÈtelmaus (Clethrionomys glareolus) unter Einfluû hormonaler und sozialer Faktoren Die RoÈtelmaus ist ein weit verbreitetes europaÈisches Nagetier. In natuÈrlichen Populationen zeigen erwachsene Weibchen TerritorialitaÈt, und waÈhrend der Brunstzeit uÈberlappt das Revier jeden Weibchens das mehrerer MaÈnnchen. Ziel dieser Studie war die Untersuchung des Einflusses der sexuellen Erfahrung von Weibchen und MaÈnnchen auf das Paarungsverhalten. Die Ergebnisse zeigen, daû die AktivitaÈt der Weibchen durch deren sexuelle Erfahrung und durch die des Partners modifiziert war.
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Die MaÈnnchen zeigten auch groÈûere AktivitaÈt in der Gegenwart des einen virginellen Weibchens als in der von erfahrenen Weibchen. Weibchen in post-partum Ústrus waren aktiver als virginelle Weibchen, und Gonadotropin-Injektionen wirkten auf die virginellen Weibchen stimulierend. MaÈnnchen zeigten in Anwesenheit von jungfraÈulichen Weibchen, die mit Gonadotropin-Injektionen behandelt waren und gegenuÈber post-partum-Ústrus-Weibchen aÈhnliche sexuelle AktivitaÈt. Sexuelle Erfahrung verstaÈrkte die AktivitaÈt der MaÈnnchen. Sie naÈherten sich haÈufig den rezeptiven Weibchen und zeigten mehr Kopulationsversuche als unerfahrene MaÈnnchen. HoÈhere AktivitaÈt eines erfahrenen Partners kann moÈglicherweise die Kopulation mit einem anderen MaÈnnchen waÈhrend ein und desselben Ústrus verhindern, wodurch der Nachwuchs nur von einem MaÈnnchen erzeugt wird. Die vorliegenden Ergebnisse zeigen, daû Bulbektomie, nicht aber Vomeronasalektomie bei sexuell erfahrenen MaÈnnchen die sexuelle AktivitaÈt verminderte. Nur 2 von 6 der LaÈsion unterzogenen MaÈnnchen kopulierten mit rezeptiven Weibchen.
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Authors' addresses: Anna Marchlewska-Koj, Maøgorzata Kruczek, Institute of Environmental Sciences, Jagiellonian University, ul. Ingardena 6, 30-060 KrakoÂw, Poland (e-mail: [email protected]); Paweø Olejniczak, Institute of Nature Conservation, Polish Academy of Sciences, Mickiewicza 33, 31-120 KrakoÂw, Poland