Behavioural Processes 43 (1998) 79 – 86
Female prairie voles (Microtus ochrogaster) fail to form a new pair after loss of mate Theresa Pizzuto a, Lowell L. Getz b,* b
a Department of Entomology, Clemson Uni6ersity, Clemson, SC 29634, USA Department of Ecology, Ethology and E6olution, Uni6ersity of Illinois, Urbana, IL 61821, USA
Received 25 June 1997; received in revised form 11 November 1997; accepted 13 November 1997
Abstract We tested experimentally the hypothesis that failure to acquire a new mate by monogamously paired female Microtus ochrogaster that lose their mate represents pair-bonding behavior, not a lack of available males. Males were removed from reproductive male–female pairs and the females provided an opportunity to pair with unfamiliar sexually inexperienced or experienced males in a semi-natural arena. Only three of 22 (13.6%) females formed a new pair, two with inexperienced males and one with an experienced male. Three other females spent more time with an experienced male than with the inexperienced male, but did not form a pair or co-nest with that male. The results confirm field observations that availability of males is not a factor in the failure of female prairie voles to form a new pair following loss of their mate. This study concludes failure to form a new pair is associated with pair-bonding behavior. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Prairie vole; Pair bonding; Monogamy
1. Introduction Three types of social groups have been identified in free-living populations of the prairie vole, Microtus ochrogaster: male – female pairs, single females and communal groups (Getz et al., 1990a, 1993). Male–female pairs include only one reproductive adult male and female; if offspring remain
* Corresponding author.
at the nest after becoming adults ( ] 30 days of age), the group is considered communal. Single female groups include only one reproductive adult female and no resident adult male; if offspring remain after becoming adult, the group is considered communal. The basic social organization of M. ochrogaster, consists of communal groups, formed mainly by addition of philopatric offspring to male–female pair (59.5%) or single female (19.5%) breeding units (Getz et al., 1993). Owing to high mortality
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from snakes during April to mid October, few nestlings survive to form communal groups (Getz et al., 1990b). As a result, during the spring to early autumn period 36.3% of the breeding units are male–female pairs, 36.8% are single females and 26.9% are communal. Beginning in mid October, when snakes hibernate and nestling survival increases, the proportion of communal groups increases to 68.6%, while male – female pairs comprise 24.0% and single females 7.4% of the groups (Getz et al., 1993). The breeding period of M. ochrogaster (in east-central Illinois) typically extends from early April through late November. Thus, single female breeding units are prominent in the population for much of the breeding period. Male–female pairs of M. ochrogaster display behavioral and hormonal traits typically associated with monogamy, i.e. cohabiting a nest, sharing a common home range, display of paternal behavior (huddling, grooming and retrieval of offspring (Kleiman, 1977; Carter and Getz, 1993; Getz et al., 1990a, 1993). A total of 75% of the male –female pairs of M. ochrogaster break up by death of one or both of the members of the pair. In only 10% of the dissolutions does a male abandon the female (Getz et al. 1990a). Carter et al. (1995) provide evidence for pair-bonding in M. ochrogaster, i.e. preference for contact with a familiar sexual partner, intrasexual aggression towards unfamiliar conspecifics (mate guarding), and resource sharing. Further, increased levels of corticosterone were recorded when an animal was separated from its mate, or placed with an unfamiliar partner. Carter et al. (1995) also demonstrated the role of oxytocin, vasopressin and corticosterone in the neuroregulation of pairbonding in M. ochrogaster. Williams et al. (1992) found that male–female pairs of M. ochrogaster pair-bonded after 6 h of mating. Moehlman (1983) and Lorenz (1991) have indicated that animals displaying strong pair bonds may not form a new pair following loss of their mate. This appears possible for M. ochrogaster; only 19.1% of the female and 19.4% of the male survivors of a male– female pair formed a permanent pair-bond with a new mate (Getz et al., 1993). Most of the single female breeding units
represented survivors of male–female pair breakups (Getz et al., 1993). When a new male–female pair formed, the new male mate moved into the nest of the female and shared her home range. Females do not move in with a male. Thus, pair formation is not a function of the female selecting a male with a high quality territory. Males forming the pair were not settled into a home range or territory until they moved in with the female. Of those females that did form a new pair, the mean time from loss of the mate to formation of a new pair was 5.0+ 1.2 days. Failure to acquire a new mate does not appear to be a result of unavailability of unpaired adult males; approximately 46% of the adult males in the population during the breeding period are wanderers, rather than residents of a social group (Getz et al., 1993). During the breeding period, there are usually at least three times as many unpaired males as single females in the population at any given time (Getz and McGuire, unpublished). The unsettled males make frequent visits to the nest and periphery of the home range of established social groups, including single females (McGuire et al., 1990a,b). Those single females that do not acquire a new mate remain reproductive. They copulate with wandering males that do not move in with the female or share her home range. There is no indication that the same male sires subsequent litters of a given single female. All females which had been members of a male–female pair were sexually experienced. All were either pregnant or had produced at least one litter by their original mate prior to dissolution of the pair. Since paired females in postpartum estrus typically copulate within minutes of giving birth, many not only would be lactating, but also would be pregnant when they lost their mate. Of the 12 females that acquired a new mate, six were lactating when the male moved into her nest, while four were both pregnant and lactating. Of the two instances of the female being nonreproductive when the new pair formed, one was in February when the population was nonreproductive. Of those wandering males for which the reproductive histories were known, approximately 90% most likely were sexually experienced (Getz and McGuire, unpublished). However, there was
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no way of knowing if they had participated in rearing a litter to weaning. Female M. ochrogaster achieve first estrus through acquisition (by naso-genital sniffing) of an ‘activation’ chemosignal present in the urine of males (Carter et al., 1980). Females remain in postpartum estrus for no longer than 48 h (Hofmann and Getz, 1986); females not becoming pregnant during this time must acquire the activation chemosignal to come into estrus again. One would anticipate, apriori, that female survivors of pair dissolutions would display a preference for experienced males when pairing with a new male (Huck and Banks, 1982a,b). In those few instances where the female was not in postpartum estrus, experienced males would be presumed to respond differently to the female, resulting in more rapid reproductive activation of the female, than would inexperienced males. If so, females pairing with experienced males should produce litters sooner than when pairing with inexperienced males. However, there is no evidence that there are behavioral differences between inexperienced and experienced M. ochrogaster males in respect to reproductive activation of females or that female M. ochrogaster can make such a distinction when pairing or mating with a male (Witt et al., 1988). If single females should display a preference for pairing with inexperienced males, such behavior might explain why so few female survivors of pair break-ups form a new pair. Field data are not adequate to test the above assumptions. We therefore conducted a laboratory study to test the hypotheses that: (1) experienced females would produce litters sooner when paired with experienced males than with inexperienced males; (2) few female survivors of a male – female pair dissolution would form a new pair, but if they did, would do so with an experienced male; (3) if not forming a pair, female survivors would display a preference for association with experienced males rather than with inexperienced males. If none of these hypotheses are supported, the experimental results would be construed as another indication of pair-bonding in M. ochrogaster (Moehlman, 1983; Lorenz, 1991).
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2. Materials and methods Prairie voles used in this study were first, second, or third generation offspring of voles trapped near Urbana, IL. The voles were housed in 24× 48× 20 cm plastic cages which contained a peat moss–wood chip substrate and a 10–20 cm layer of straw. Sunflower seeds, oats, Purina rabbit chow, cracked corn and water were provided ad libitum. The animals were maintained in a room with a 15L:9D photoperiod (light onset at 2000 h); temperatures ranged from 20–25°C.
2.1. Reproducti6e success There is no evidence available to indicate differential reproductive effectiveness of reproductively experienced and reproductively inexperienced male M. ochrogaster when forming a new pair. We therefore conducted an experiment to examine this question. We used as an indication of ‘effectiveness’ the length of time from first association of a male with an experienced female until a litter was produced and the litter size resulting from a pairing. We used experienced females to replicate field conditions regarding a female survivor of a pair acquiring a new mate. As indicated above, most such female survivors would be pregnant and lactating, or both, when they lost their mate. However, to standardize conditions between trials, we used as test females only those that had been separated from their mate before giving birth. The females had reared two litters and their original mate removed prior to the birth of the second litter. The females were placed in a trial the day after the second litter was weaned. Therefore, the females were separated from their original mate for at least 23 days. The females would not be in estrus when paired with a male; if they do not copulate with a male within 48 hours of giving birth, most females go out of postpartum estrus (Hofmann and Getz, 1986). In this way we were assured that the test male had activated and copulated with the female and that the resulting litter was his. Experienced males had produced a litter and remained with the female until the young were
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weaned. The male was then removed and kept in a separate cage at least 10 days before being paired with a female. Inexperienced males were separated from their parents at 21 days of age and maintained in same-sex sibling groups until paired in the experiment. Ages of the males and females when placed in the experiment ranged from 70 – 90 days; similar aged animals were paired where possible. The cages were examined daily to record when birth had occurred. Days from pairing to birth of the litter and litter size were recorded.
2.2. Association of females and males 2.2.1. Experimental animals In these experiments an attempt was made to: (1) replicate conditions observed in the field in respect to reproductive condition of the female when she lost her mate; and (2) to match up as closely as possible the ages of the two test males used in each trial. Three female groups were utilized: (1) lactating, not pregnant, in estrus; (2) lactating, most likely in very early pregnancy; and (3) lactating and in mid pregnancy. All experienced males had been paired with a female and had successfully reared a litter. These males would represent those males that had been previously paired, but had lost their mate. The inexperienced males were representative of natal dispersers that were forming their first pair. Experienced females were obtained by pairing the female with a male of approximately the same age (40–80 days of age) and allowing them to produce and rear a litter together. These females would have been pair-bonded with the male (Williams et al., 1992). Males were removed 1 day prior to, 5 days following, or 13 days following birth of the next litter, depending upon the trial. Experienced males were obtained by pairing a male at least 3 months old with a female of similar age. Only those males fathering a litter were used in the trials. After the litter was 21 days old, the adult male was removed and placed in a separate cage for 3– 4 weeks before being used in a trial, thus, representing animals that were reproductively experienced, but had been wandering for some time, as had most males in the field, before pairing with a single female. Inexperienced males
were removed from the parents at 21 days of age and housed with same-sex siblings. All males were approximately 4 months old when used in a trial. The females ranged from 2–4 months of age when used in the experiment.
2.2.2. Arenas Behavioral observations were conducted in six 1.5× 2.5 m2 Plexiglas-bottomed arenas (Lyons and Getz, 1993) located in the same room in which the breeding pairs were housed. The arenas had a 45×65 cm2 chamber at each end, at opposite corners. The walls of the arenas and chambers consisted of 50 cm aluminum flashing nailed on the inside of 20 cm wooden sides. Two 2.0 cm holes connected each chamber with the central portion (center) of the arena. A 3-cm layer of damp peat moss was placed on the floor of the arena and compartments and 20 cm of wheat straw was placed over the peat moss. A water bottle was suspended in each compartment and in the center of the arena. The same food mixture used in the breeding colony was provided ad libitum in the compartments and center of the arena. The peat moss and straw were replaced following each trial and the Plexiglas and aluminum flashing washed with warm water and soap. Inexperienced and experienced males were assigned to the end compartments of an arena on a rotating schedule among trials. 2.2.3. Obser6ational protocol The female and her litter were placed in the center of the arena on the day they were separated from the male. At 1 day following introduction of the female into the arena, an experienced male was placed in one chamber and an inexperienced male in the other chamber. The males were provided with neck collars made of plastic cable ties that prevented them from passing through the openings of the chambers into the center of the arena. The female had free movement through the openings into both chambers. Before being placed in the arena, Nyanzol dye was applied to the belly fur of each animal for individual identification. Observations began 1 day following introduction of the female into the arena. This allowed time for her to form runways through the peat
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moss, the bottoms of which were open on the Plexiglas. All observations were made from under the arenas, with the observer lying on an auto mechanic ‘car creeper’. Three electronic timers were used to record: (1) the amount of time the female spent in each part of the arena — center, chamber of the inexperienced male and chamber of the experienced male; (2) amount of time in the nest with her young (a note was made as to the location of the nests, center or in a given chamber); and (3) moving around in the runways (and location of the runways, in the center or in a chamber). A record was made of each approach the female made to the openings of the chambers (stuck her nose into the opening) and whether or not the female entered the chamber or withdrew. We recorded the number of social interactions between the female and each of the males and those which were aggressive during each trial. An approach to within 1 cm of the male was considered an interaction. ‘Aggressive’ interactions included threats (the vole raises its forefeet from the floor, extents its head towards the other animal and bares its teeth), lunges (the vole thrusts its head towards the other animal or jumps at the animal; may or may not bite the other animal), boxing (the vole assumes an upright position and strikes at the other animal with its forefeet), chasing (one vole pursues the other), chattering (rapid squeaking noise emitted with the head directed at the other animal), and biting the other animal (Hofmann et al., 1982). Observations of the behavior of the female were recorded for two 30-min periods each day, early to mid morning (08:00 – 11:00 h) and early to late evening (20:00–23:00 h) for 5 days. In addition, one 30-min ‘scan’ observation was made of the males each day (15 min for each male) approximately midway between the two routine observations. These scan observations were used to obtain additional data regarding location of the female in relation to the two males. The amount of time the female spent in the chamber of each male was recorded during the scans.
2.2.4. Trials A total of 24 females were tested, eight each using females separated from their mate, either
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the day before birth of the young (Group 1), 5 days after birth (Group 2) or 13 days after birth (Group 3). These represented situations in which the female had lost her mate just before giving birth, soon after giving birth, and 10 days before she would have given birth again (she most likely had mated again in the latter two groups). In two trials (one each in trials when the males were removed 1 day prior to and 5 days following the female giving birth) both males died of injuries (presumably inflicted by the female) during the 2nd day of observations; these trials were not used in the analyses.
2.2.5. Controls A total of 12 control females (four in each of the three treatments) were placed in the arenas on the same schedule. Males were not placed in the end chambers. The same observational protocol was followed as for when males were in the chambers, except that no female–male interaction data were possible. Trials of the different groups and the controls were run concurrently throughout the course of the study. 2.2.6. Data analyses The following criteria were established to determine if a female had paired with a male: (1) by the termination of the 5-day trial, the female and her young had moved into the compartment of the male, were co-nesting with him, and had been nesting there for at least the last four 30-min observational periods; (2) the female spent as much time in the compartment of the ‘paired’ male as in the other two locations combined; and (3) the female spent at least twice as much time in the compartment of the ‘paired’ male as in the compartment of the other male. The data were also analyzed to determine if the female appeared to prefer to associate with one male over another, if she did not pair with one of the males. To display such an association for a given male, the female had to: (1) spend more than twice the amount of time in the compartment with the ‘preferred’ male as with the other male and this had to occur for at least five of the ten observational periods; and (2) spend a minimum total of
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Table 1 Responses of female Microtus ochrogaster that have been separated from their mates to inexperienced and experienced males Group
1 (7) 2 (7) 3 (8) Total (22)
Paired
Associated Inexp.
Entered chamber
Aggressive interactions
Exp.
Inexp. (%)
Exp. (%)
Inexp. (%)
Exp. (%)
61.7 72.4 42.9 60.5
46.3 74.0 54.9 57.3
74.6 78.7 62.0 71.2
57.7 53.0 32.4 42.3
Inexp.
Exp.
1 1
1
1
2
1
2 3
(115) (156) (114) (385)
(134) (104) (111) (349)
(118) (61) (92) (271)
(78) (83) (111) (272)
Values in parens are sample sizes. Group 1, separated 1 day before birth of litter; Group 2, separated 5 days postpartum; Group 3, separated 13 days postpartum. See text for definitions of responses.
60 minutes in the chamber of the ‘preferred’ male during the ten observational periods. Data were analyzed using t-tests and x 2-tests of association.
3. Results and discussion There was no difference in the reproductive success of experienced females mated to inexperienced or experienced males in respect to time to birth of the litter or litter size (time to birth of first litter: inexperienced males, 23.3+ 0.6 days; experienced males, 22.5+ 0.6 days; t = 1.55, df = 1, P\0.3. Average litter size: inexperienced males, 5.3+0.3; experienced males, 4.8+0.4; t = 0.88, df=1, P\0.5). There was no difference among the females separated at different days postpartum with respect to pairing with or association with inexperienced and experienced males (Table 1). The data for the three trials are therefore combined for analysis. Data for the controls were similarly combined. In only three of the 22 (13.6%) trials used in the analyses did the female meet the criteria for forming a pair with one of the males. Two of the females paired with inexperienced males, the other with an experienced male. These females moved their young into the nest they shared with the male. Three females displayed a preference for association with the experienced male (Table 1); none was observed in the nest of that male, however. The pups of all females placed in the
arena 13 days postpartum wandered in an out of the chamber of the males, but did not remain in their nests. Females that nested in the center of the arena (n=17) spent 78.3 + 2.8% of the total time in that nest. Four females, while spending the greatest proportion of the total time in their own nest in the center, were observed briefly (19.7–27.0% of the time) in the nest of a male. None met the criteria for pairing with or associating with a given male. One female did not nest in the center, but moved back and forth between the nests of the two males (percent of total time in nests: experienced male, 23.8%; inexperienced male, 19.7%). Control females (n= 12) spent 78.2+ 3.3% of their time in the nest. All but one control female nested exclusively in the center of the arena. The latter female used a nest in one end compartment for the first 3 days and then a nest in the other end compartment for the last 2 days. The scan trials data conformed to the detailed observations regarding the pairing or associating of a female with a given male. The proportion of total encounters between a female and an inexperienced male that were aggressive were greater than were those between the female and an experienced male (271/193, 71.2% and 272/115, 42.3%, respectively; x 2 = 19.75, df= 1, PB0.001). The total approaches of the female to the entry openings and the proportion of these approaches which resulted in subsequent entry into the inexperienced male and experienced male compartments did not differ (inexperienced, 349/ 200, 57.3%; experienced, 385/233, 60.5%, ap-
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proaches and entry, respectively; x 2 =0.386, df= l, P\0.50). Comparable data for the controls (total approaches to either chamber opening and the number of times the female went into the vacant chamber) were 170/109, 64.1%. Offspring of females first put in the arena 13 days postpartum were already active out of the nest. They explored all parts of the arena including the compartments of both males, irrespective of where the female nested. Offspring of females placed in the arena 1 day and 5 days postpartum were routinely observed being taken (either attached to the nipples or carried in the mouth of the female) into the compartments of the males when the female went in even for short visits. They were taken into the nest of the male when the female went into his nest. A total of 128 offspring were exposed to at least one male during the study. Only one instance of infant mortality from injury was observed in all the trials. Four of the six young of a female (non paired) in the 5th day postpartum separation trials were found dead with bite marks to the head in the inexperienced male compartment. We did not observe which animal, male or female, killed the young. In another trial (5-day postpartum trial; non paired) one young disappeared during the trial. All 53 of the young in the control trials survived the 5-day observation period. In trials in which the young were taken to the nest of the male by the female, or entered the male chamber on their own, the males displayed paternal behavior, including huddling, grooming and retrieving.
Aggressive interactions were more common between females and inexperienced males than between females and experienced males. Infanticide was minimal and did not appear to be a factor in failure of females to form a new pair. Further, there was no difference in the attraction to or avoidance of inexperienced or experienced males with respect to the female entering the chamber of a male. These observations are consistent with data demonstrating that experienced females paired with inexperienced males produced litters as soon as those paired with experienced males. The results of this experimental study support the assumption that members of breeding pairs of prairie vole pairs are pair-bonded (Carter et al., 1995). We conclude that females that lose their mates most likely remain as single females as a result of behavior associated with pair-bonding. That there are few inexperienced males in the population is not a factor in the females remaining unpaired.
4. Conclusions
References
Consistent with field observations on female M. ochrogaster that lost their mates, only 13.6% females in this study formed a new pair. Those that did pair again did not display a preference for inexperienced or experienced males. Failure to form a new pair in this study obviously was not a result of unavailability of a male as two males were available within the arena. Previous sexual experience by the male was not a factor in forming a new pair; both sexually inexperienced and experienced males were available to each female.
Carter, C.S., Getz, L.L., Gavish, L., McDermott, J.L., Arnold, P., 1980. Male-related pheromones and the activation of female reproduction in the prairie vole (Microtus ochrogaster). Biol. Reprod. 23, 1038 – 1045. Carter, C.S., Getz, L.L., 1993. Monogamy and the prairie vole. Sci. Am. 268, 100 – 106. Carter, C.S., DeVries, A.C., Getz, L.L., 1995. Physiological substrates of mammalian monogamy: the prairie vole model. Neurosci. Biobehav. Rev. 19, 303 – 314. Getz, L.L., McGuire, B., Hofmann, J., Pizzuto, T., Frase, B., 1990a. Social organization and mating system of the prairie vole, Microtus ochrogaster. In: Tamarin, R.H., Ostfeld, R.S., Pugh, S.R., Bujalska, G. (Eds.), Social systems
Acknowledgements This study was supported in part by NIH Grant HD 09328 to LLG. We thank Betty McGuire for her invaluable assistance throughout this study, including help with the observations. We also thank Leonard Clay, Cheryl Fikejs, Peggy Gronemeyer, Paul Klatt, Carolyn Roselle, Mike Unger and Esmond Yen for their assistance with the observations and colony maintenance.
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and population cycles in voles. Birkhauser Verlag, Basel, pp. 69 – 80. Getz, L.L., Solomon, N.G., Pizzuto, T., 1990b. The effects of predation of snakes on social organization in the prairie vole, Microtus ochrogaster. Am. Midl. Nat. 123, 365–371. Getz, L.L., McGuire, B., Hofmann, J., Pizzuto, T., Frase, B., 1993. Social organization of the prairie vole, Microtus ochrogaster. J. Mammal. 74, 44–58. Hofmann, J.E., Getz, L.L., Klatt, B.J., 1982. Levels of male aggressiveness in fluctuating populations of Microtus ochrogaster and M. pennsyl6anicus. Can. J. Zool. 60, 898– 912. Hofmann, J.E., Getz, L.L., 1986. Duration of postpartum estrus in the prairie vole. Bull. Psychnom. Soc. 24, 300– 301. Huck, U.W., Banks, E.M., 1982a. Differential attraction of females to dominant males: olfactory discrimination and mating preference in the brown lemming (Lemmus trimucronatus). Behav. Ecol. Soc. Biol. 11, 217–222. Huck, U.W., Banks, E.M., 1982b. Male dominance status, female choice and mating success in the brown lemming (Lemmus trimucronatus). An. Behav. 30, 665–675. Kleiman, D.G., 1977. Monogamy in mammals. Q. Rev. Biol. 52, 39 – 69. Lorenz, K., 1991. Here am I—Where are you? The behavior of the Greylag Goose. Harcourt Brace Jovanovich, New York, p. 270.
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Lyons, S.A., Getz, L.L., 1993. Reproductive activation of virgin female prairie voles (Microtus ochrogaster) by paired and unpaired males. Behav. Process. 29, 191 – 200. McGuire, B., Pizzuto, T., Getz, L.L., 1990a. Potential for social interaction in a natural population of prairie voles (Microtus ochrogaster). Can. J. Zool. 68, 391 – 398. McGuire, B., Pizzuto, T., Getz, L.L., 1990b. Patterns of visitation in prairie voles (Microtus ochrogaster) reveal a role for males in population regulation. In: Tamarin, R.H., Ostfeld, R.S., Pugh, S.R., Bujalska, G. (Eds.), Social systems and population cycles in voles. Birkhauser Verlag, Basel, pp. 89 – 99. Moehlman, P.D., 1983. Socioecology of silverbacked and golden jackals (Canis mesomelas and Canis aureus). In: Eisenberg, J.F., Kleiman, D.G. (Eds.), Advances in the study of mammalian behavior. Am. Soc. Mammal. Spec. 7, 432 – 453. Williams, J.R., Catania, K.C., Carter, C.S., 1992. Development of partner preferences in female prairie voles (Microtus ochrogaster): the role of social and sexual experience. Horm. Behav. 26, 339 – 349. Witt, D.M., Carter, C.S., Carlstead, K., Read, L.D., 1988. Sexual and social interactions preceding and during maleinduced oestrous in prairie voles, Microtus ochrogaster. An. Behav. 36, 1465 – 1471.
Female prairie voles (Microtus ochrogaster) fail to form a new pair after loss of mate Theresa Pizzuto a, Lowell L. Getz b,* b
a Department of Entomology, Clemson Uni6ersity, Clemson, SC 29634, USA Department of Ecology, Ethology and E6olution, Uni6ersity of Illinois, Urbana, IL 61821, USA
Received 25 June 1997; received in revised form 11 November 1997; accepted 13 November 1997
Abstract We tested experimentally the hypothesis that failure to acquire a new mate by monogamously paired female Microtus ochrogaster that lose their mate represents pair-bonding behavior, not a lack of available males. Males were removed from reproductive male–female pairs and the females provided an opportunity to pair with unfamiliar sexually inexperienced or experienced males in a semi-natural arena. Only three of 22 (13.6%) females formed a new pair, two with inexperienced males and one with an experienced male. Three other females spent more time with an experienced male than with the inexperienced male, but did not form a pair or co-nest with that male. The results confirm field observations that availability of males is not a factor in the failure of female prairie voles to form a new pair following loss of their mate. This study concludes failure to form a new pair is associated with pair-bonding behavior. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Prairie vole; Pair bonding; Monogamy
1. Introduction Three types of social groups have been identified in free-living populations of the prairie vole, Microtus ochrogaster: male – female pairs, single females and communal groups (Getz et al., 1990a, 1993). Male–female pairs include only one reproductive adult male and female; if offspring remain
* Corresponding author.
at the nest after becoming adults ( ] 30 days of age), the group is considered communal. Single female groups include only one reproductive adult female and no resident adult male; if offspring remain after becoming adult, the group is considered communal. The basic social organization of M. ochrogaster, consists of communal groups, formed mainly by addition of philopatric offspring to male–female pair (59.5%) or single female (19.5%) breeding units (Getz et al., 1993). Owing to high mortality
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from snakes during April to mid October, few nestlings survive to form communal groups (Getz et al., 1990b). As a result, during the spring to early autumn period 36.3% of the breeding units are male–female pairs, 36.8% are single females and 26.9% are communal. Beginning in mid October, when snakes hibernate and nestling survival increases, the proportion of communal groups increases to 68.6%, while male – female pairs comprise 24.0% and single females 7.4% of the groups (Getz et al., 1993). The breeding period of M. ochrogaster (in east-central Illinois) typically extends from early April through late November. Thus, single female breeding units are prominent in the population for much of the breeding period. Male–female pairs of M. ochrogaster display behavioral and hormonal traits typically associated with monogamy, i.e. cohabiting a nest, sharing a common home range, display of paternal behavior (huddling, grooming and retrieval of offspring (Kleiman, 1977; Carter and Getz, 1993; Getz et al., 1990a, 1993). A total of 75% of the male –female pairs of M. ochrogaster break up by death of one or both of the members of the pair. In only 10% of the dissolutions does a male abandon the female (Getz et al. 1990a). Carter et al. (1995) provide evidence for pair-bonding in M. ochrogaster, i.e. preference for contact with a familiar sexual partner, intrasexual aggression towards unfamiliar conspecifics (mate guarding), and resource sharing. Further, increased levels of corticosterone were recorded when an animal was separated from its mate, or placed with an unfamiliar partner. Carter et al. (1995) also demonstrated the role of oxytocin, vasopressin and corticosterone in the neuroregulation of pairbonding in M. ochrogaster. Williams et al. (1992) found that male–female pairs of M. ochrogaster pair-bonded after 6 h of mating. Moehlman (1983) and Lorenz (1991) have indicated that animals displaying strong pair bonds may not form a new pair following loss of their mate. This appears possible for M. ochrogaster; only 19.1% of the female and 19.4% of the male survivors of a male– female pair formed a permanent pair-bond with a new mate (Getz et al., 1993). Most of the single female breeding units
represented survivors of male–female pair breakups (Getz et al., 1993). When a new male–female pair formed, the new male mate moved into the nest of the female and shared her home range. Females do not move in with a male. Thus, pair formation is not a function of the female selecting a male with a high quality territory. Males forming the pair were not settled into a home range or territory until they moved in with the female. Of those females that did form a new pair, the mean time from loss of the mate to formation of a new pair was 5.0+ 1.2 days. Failure to acquire a new mate does not appear to be a result of unavailability of unpaired adult males; approximately 46% of the adult males in the population during the breeding period are wanderers, rather than residents of a social group (Getz et al., 1993). During the breeding period, there are usually at least three times as many unpaired males as single females in the population at any given time (Getz and McGuire, unpublished). The unsettled males make frequent visits to the nest and periphery of the home range of established social groups, including single females (McGuire et al., 1990a,b). Those single females that do not acquire a new mate remain reproductive. They copulate with wandering males that do not move in with the female or share her home range. There is no indication that the same male sires subsequent litters of a given single female. All females which had been members of a male–female pair were sexually experienced. All were either pregnant or had produced at least one litter by their original mate prior to dissolution of the pair. Since paired females in postpartum estrus typically copulate within minutes of giving birth, many not only would be lactating, but also would be pregnant when they lost their mate. Of the 12 females that acquired a new mate, six were lactating when the male moved into her nest, while four were both pregnant and lactating. Of the two instances of the female being nonreproductive when the new pair formed, one was in February when the population was nonreproductive. Of those wandering males for which the reproductive histories were known, approximately 90% most likely were sexually experienced (Getz and McGuire, unpublished). However, there was
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no way of knowing if they had participated in rearing a litter to weaning. Female M. ochrogaster achieve first estrus through acquisition (by naso-genital sniffing) of an ‘activation’ chemosignal present in the urine of males (Carter et al., 1980). Females remain in postpartum estrus for no longer than 48 h (Hofmann and Getz, 1986); females not becoming pregnant during this time must acquire the activation chemosignal to come into estrus again. One would anticipate, apriori, that female survivors of pair dissolutions would display a preference for experienced males when pairing with a new male (Huck and Banks, 1982a,b). In those few instances where the female was not in postpartum estrus, experienced males would be presumed to respond differently to the female, resulting in more rapid reproductive activation of the female, than would inexperienced males. If so, females pairing with experienced males should produce litters sooner than when pairing with inexperienced males. However, there is no evidence that there are behavioral differences between inexperienced and experienced M. ochrogaster males in respect to reproductive activation of females or that female M. ochrogaster can make such a distinction when pairing or mating with a male (Witt et al., 1988). If single females should display a preference for pairing with inexperienced males, such behavior might explain why so few female survivors of pair break-ups form a new pair. Field data are not adequate to test the above assumptions. We therefore conducted a laboratory study to test the hypotheses that: (1) experienced females would produce litters sooner when paired with experienced males than with inexperienced males; (2) few female survivors of a male – female pair dissolution would form a new pair, but if they did, would do so with an experienced male; (3) if not forming a pair, female survivors would display a preference for association with experienced males rather than with inexperienced males. If none of these hypotheses are supported, the experimental results would be construed as another indication of pair-bonding in M. ochrogaster (Moehlman, 1983; Lorenz, 1991).
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2. Materials and methods Prairie voles used in this study were first, second, or third generation offspring of voles trapped near Urbana, IL. The voles were housed in 24× 48× 20 cm plastic cages which contained a peat moss–wood chip substrate and a 10–20 cm layer of straw. Sunflower seeds, oats, Purina rabbit chow, cracked corn and water were provided ad libitum. The animals were maintained in a room with a 15L:9D photoperiod (light onset at 2000 h); temperatures ranged from 20–25°C.
2.1. Reproducti6e success There is no evidence available to indicate differential reproductive effectiveness of reproductively experienced and reproductively inexperienced male M. ochrogaster when forming a new pair. We therefore conducted an experiment to examine this question. We used as an indication of ‘effectiveness’ the length of time from first association of a male with an experienced female until a litter was produced and the litter size resulting from a pairing. We used experienced females to replicate field conditions regarding a female survivor of a pair acquiring a new mate. As indicated above, most such female survivors would be pregnant and lactating, or both, when they lost their mate. However, to standardize conditions between trials, we used as test females only those that had been separated from their mate before giving birth. The females had reared two litters and their original mate removed prior to the birth of the second litter. The females were placed in a trial the day after the second litter was weaned. Therefore, the females were separated from their original mate for at least 23 days. The females would not be in estrus when paired with a male; if they do not copulate with a male within 48 hours of giving birth, most females go out of postpartum estrus (Hofmann and Getz, 1986). In this way we were assured that the test male had activated and copulated with the female and that the resulting litter was his. Experienced males had produced a litter and remained with the female until the young were
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weaned. The male was then removed and kept in a separate cage at least 10 days before being paired with a female. Inexperienced males were separated from their parents at 21 days of age and maintained in same-sex sibling groups until paired in the experiment. Ages of the males and females when placed in the experiment ranged from 70 – 90 days; similar aged animals were paired where possible. The cages were examined daily to record when birth had occurred. Days from pairing to birth of the litter and litter size were recorded.
2.2. Association of females and males 2.2.1. Experimental animals In these experiments an attempt was made to: (1) replicate conditions observed in the field in respect to reproductive condition of the female when she lost her mate; and (2) to match up as closely as possible the ages of the two test males used in each trial. Three female groups were utilized: (1) lactating, not pregnant, in estrus; (2) lactating, most likely in very early pregnancy; and (3) lactating and in mid pregnancy. All experienced males had been paired with a female and had successfully reared a litter. These males would represent those males that had been previously paired, but had lost their mate. The inexperienced males were representative of natal dispersers that were forming their first pair. Experienced females were obtained by pairing the female with a male of approximately the same age (40–80 days of age) and allowing them to produce and rear a litter together. These females would have been pair-bonded with the male (Williams et al., 1992). Males were removed 1 day prior to, 5 days following, or 13 days following birth of the next litter, depending upon the trial. Experienced males were obtained by pairing a male at least 3 months old with a female of similar age. Only those males fathering a litter were used in the trials. After the litter was 21 days old, the adult male was removed and placed in a separate cage for 3– 4 weeks before being used in a trial, thus, representing animals that were reproductively experienced, but had been wandering for some time, as had most males in the field, before pairing with a single female. Inexperienced males
were removed from the parents at 21 days of age and housed with same-sex siblings. All males were approximately 4 months old when used in a trial. The females ranged from 2–4 months of age when used in the experiment.
2.2.2. Arenas Behavioral observations were conducted in six 1.5× 2.5 m2 Plexiglas-bottomed arenas (Lyons and Getz, 1993) located in the same room in which the breeding pairs were housed. The arenas had a 45×65 cm2 chamber at each end, at opposite corners. The walls of the arenas and chambers consisted of 50 cm aluminum flashing nailed on the inside of 20 cm wooden sides. Two 2.0 cm holes connected each chamber with the central portion (center) of the arena. A 3-cm layer of damp peat moss was placed on the floor of the arena and compartments and 20 cm of wheat straw was placed over the peat moss. A water bottle was suspended in each compartment and in the center of the arena. The same food mixture used in the breeding colony was provided ad libitum in the compartments and center of the arena. The peat moss and straw were replaced following each trial and the Plexiglas and aluminum flashing washed with warm water and soap. Inexperienced and experienced males were assigned to the end compartments of an arena on a rotating schedule among trials. 2.2.3. Obser6ational protocol The female and her litter were placed in the center of the arena on the day they were separated from the male. At 1 day following introduction of the female into the arena, an experienced male was placed in one chamber and an inexperienced male in the other chamber. The males were provided with neck collars made of plastic cable ties that prevented them from passing through the openings of the chambers into the center of the arena. The female had free movement through the openings into both chambers. Before being placed in the arena, Nyanzol dye was applied to the belly fur of each animal for individual identification. Observations began 1 day following introduction of the female into the arena. This allowed time for her to form runways through the peat
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moss, the bottoms of which were open on the Plexiglas. All observations were made from under the arenas, with the observer lying on an auto mechanic ‘car creeper’. Three electronic timers were used to record: (1) the amount of time the female spent in each part of the arena — center, chamber of the inexperienced male and chamber of the experienced male; (2) amount of time in the nest with her young (a note was made as to the location of the nests, center or in a given chamber); and (3) moving around in the runways (and location of the runways, in the center or in a chamber). A record was made of each approach the female made to the openings of the chambers (stuck her nose into the opening) and whether or not the female entered the chamber or withdrew. We recorded the number of social interactions between the female and each of the males and those which were aggressive during each trial. An approach to within 1 cm of the male was considered an interaction. ‘Aggressive’ interactions included threats (the vole raises its forefeet from the floor, extents its head towards the other animal and bares its teeth), lunges (the vole thrusts its head towards the other animal or jumps at the animal; may or may not bite the other animal), boxing (the vole assumes an upright position and strikes at the other animal with its forefeet), chasing (one vole pursues the other), chattering (rapid squeaking noise emitted with the head directed at the other animal), and biting the other animal (Hofmann et al., 1982). Observations of the behavior of the female were recorded for two 30-min periods each day, early to mid morning (08:00 – 11:00 h) and early to late evening (20:00–23:00 h) for 5 days. In addition, one 30-min ‘scan’ observation was made of the males each day (15 min for each male) approximately midway between the two routine observations. These scan observations were used to obtain additional data regarding location of the female in relation to the two males. The amount of time the female spent in the chamber of each male was recorded during the scans.
2.2.4. Trials A total of 24 females were tested, eight each using females separated from their mate, either
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the day before birth of the young (Group 1), 5 days after birth (Group 2) or 13 days after birth (Group 3). These represented situations in which the female had lost her mate just before giving birth, soon after giving birth, and 10 days before she would have given birth again (she most likely had mated again in the latter two groups). In two trials (one each in trials when the males were removed 1 day prior to and 5 days following the female giving birth) both males died of injuries (presumably inflicted by the female) during the 2nd day of observations; these trials were not used in the analyses.
2.2.5. Controls A total of 12 control females (four in each of the three treatments) were placed in the arenas on the same schedule. Males were not placed in the end chambers. The same observational protocol was followed as for when males were in the chambers, except that no female–male interaction data were possible. Trials of the different groups and the controls were run concurrently throughout the course of the study. 2.2.6. Data analyses The following criteria were established to determine if a female had paired with a male: (1) by the termination of the 5-day trial, the female and her young had moved into the compartment of the male, were co-nesting with him, and had been nesting there for at least the last four 30-min observational periods; (2) the female spent as much time in the compartment of the ‘paired’ male as in the other two locations combined; and (3) the female spent at least twice as much time in the compartment of the ‘paired’ male as in the compartment of the other male. The data were also analyzed to determine if the female appeared to prefer to associate with one male over another, if she did not pair with one of the males. To display such an association for a given male, the female had to: (1) spend more than twice the amount of time in the compartment with the ‘preferred’ male as with the other male and this had to occur for at least five of the ten observational periods; and (2) spend a minimum total of
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Table 1 Responses of female Microtus ochrogaster that have been separated from their mates to inexperienced and experienced males Group
1 (7) 2 (7) 3 (8) Total (22)
Paired
Associated Inexp.
Entered chamber
Aggressive interactions
Exp.
Inexp. (%)
Exp. (%)
Inexp. (%)
Exp. (%)
61.7 72.4 42.9 60.5
46.3 74.0 54.9 57.3
74.6 78.7 62.0 71.2
57.7 53.0 32.4 42.3
Inexp.
Exp.
1 1
1
1
2
1
2 3
(115) (156) (114) (385)
(134) (104) (111) (349)
(118) (61) (92) (271)
(78) (83) (111) (272)
Values in parens are sample sizes. Group 1, separated 1 day before birth of litter; Group 2, separated 5 days postpartum; Group 3, separated 13 days postpartum. See text for definitions of responses.
60 minutes in the chamber of the ‘preferred’ male during the ten observational periods. Data were analyzed using t-tests and x 2-tests of association.
3. Results and discussion There was no difference in the reproductive success of experienced females mated to inexperienced or experienced males in respect to time to birth of the litter or litter size (time to birth of first litter: inexperienced males, 23.3+ 0.6 days; experienced males, 22.5+ 0.6 days; t = 1.55, df = 1, P\0.3. Average litter size: inexperienced males, 5.3+0.3; experienced males, 4.8+0.4; t = 0.88, df=1, P\0.5). There was no difference among the females separated at different days postpartum with respect to pairing with or association with inexperienced and experienced males (Table 1). The data for the three trials are therefore combined for analysis. Data for the controls were similarly combined. In only three of the 22 (13.6%) trials used in the analyses did the female meet the criteria for forming a pair with one of the males. Two of the females paired with inexperienced males, the other with an experienced male. These females moved their young into the nest they shared with the male. Three females displayed a preference for association with the experienced male (Table 1); none was observed in the nest of that male, however. The pups of all females placed in the
arena 13 days postpartum wandered in an out of the chamber of the males, but did not remain in their nests. Females that nested in the center of the arena (n=17) spent 78.3 + 2.8% of the total time in that nest. Four females, while spending the greatest proportion of the total time in their own nest in the center, were observed briefly (19.7–27.0% of the time) in the nest of a male. None met the criteria for pairing with or associating with a given male. One female did not nest in the center, but moved back and forth between the nests of the two males (percent of total time in nests: experienced male, 23.8%; inexperienced male, 19.7%). Control females (n= 12) spent 78.2+ 3.3% of their time in the nest. All but one control female nested exclusively in the center of the arena. The latter female used a nest in one end compartment for the first 3 days and then a nest in the other end compartment for the last 2 days. The scan trials data conformed to the detailed observations regarding the pairing or associating of a female with a given male. The proportion of total encounters between a female and an inexperienced male that were aggressive were greater than were those between the female and an experienced male (271/193, 71.2% and 272/115, 42.3%, respectively; x 2 = 19.75, df= 1, PB0.001). The total approaches of the female to the entry openings and the proportion of these approaches which resulted in subsequent entry into the inexperienced male and experienced male compartments did not differ (inexperienced, 349/ 200, 57.3%; experienced, 385/233, 60.5%, ap-
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85
proaches and entry, respectively; x 2 =0.386, df= l, P\0.50). Comparable data for the controls (total approaches to either chamber opening and the number of times the female went into the vacant chamber) were 170/109, 64.1%. Offspring of females first put in the arena 13 days postpartum were already active out of the nest. They explored all parts of the arena including the compartments of both males, irrespective of where the female nested. Offspring of females placed in the arena 1 day and 5 days postpartum were routinely observed being taken (either attached to the nipples or carried in the mouth of the female) into the compartments of the males when the female went in even for short visits. They were taken into the nest of the male when the female went into his nest. A total of 128 offspring were exposed to at least one male during the study. Only one instance of infant mortality from injury was observed in all the trials. Four of the six young of a female (non paired) in the 5th day postpartum separation trials were found dead with bite marks to the head in the inexperienced male compartment. We did not observe which animal, male or female, killed the young. In another trial (5-day postpartum trial; non paired) one young disappeared during the trial. All 53 of the young in the control trials survived the 5-day observation period. In trials in which the young were taken to the nest of the male by the female, or entered the male chamber on their own, the males displayed paternal behavior, including huddling, grooming and retrieving.
Aggressive interactions were more common between females and inexperienced males than between females and experienced males. Infanticide was minimal and did not appear to be a factor in failure of females to form a new pair. Further, there was no difference in the attraction to or avoidance of inexperienced or experienced males with respect to the female entering the chamber of a male. These observations are consistent with data demonstrating that experienced females paired with inexperienced males produced litters as soon as those paired with experienced males. The results of this experimental study support the assumption that members of breeding pairs of prairie vole pairs are pair-bonded (Carter et al., 1995). We conclude that females that lose their mates most likely remain as single females as a result of behavior associated with pair-bonding. That there are few inexperienced males in the population is not a factor in the females remaining unpaired.
4. Conclusions
References
Consistent with field observations on female M. ochrogaster that lost their mates, only 13.6% females in this study formed a new pair. Those that did pair again did not display a preference for inexperienced or experienced males. Failure to form a new pair in this study obviously was not a result of unavailability of a male as two males were available within the arena. Previous sexual experience by the male was not a factor in forming a new pair; both sexually inexperienced and experienced males were available to each female.
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Acknowledgements This study was supported in part by NIH Grant HD 09328 to LLG. We thank Betty McGuire for her invaluable assistance throughout this study, including help with the observations. We also thank Leonard Clay, Cheryl Fikejs, Peggy Gronemeyer, Paul Klatt, Carolyn Roselle, Mike Unger and Esmond Yen for their assistance with the observations and colony maintenance.
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