Northeastern Naturalist Vol. 22, No. 3 K. McCracken, R. Lewis, and J.T. Curtis 2015 541 2015 NORTHEASTERN NATURALIST 22(3):541–550 Female-Paced Mating Does Not Affect Pair-Bond Expression by Microtus ochrogaster (Prairie Vole) Males Kelly McCracken1, Robert Lewis1, and J. Thomas Curtis1,* Abstract - Microtus ochrogaster (Prairie Vole) males typically display robust preferences for affiliation with their respective mates that indicate the expression of a pair bond. However, it recently has been shown that the strength of a male vole’s pair bond can differ depending on the reproductive status of his mate. In the present study, we examined the possibility that female-controlled pacing of the mating sequence could alter males’ affiliative behaviors in a partner-preference test by affecting reproductive success. We expected that an earlier onset of mating and pregnancy would occur if females controlled the pace of mating, which in turn, would reinforce males’ preference for their familiar mates vs. for a stranger. We found that female pacing did not affect latency to mating, mating duration, or any of the other measures of social or mating behaviors we measured. Further, femalepaced mating did not alter reproductive success as indicated by litter size. We conclude that female-paced mating in Prairie Voles does not impact the formation, consolidation, and/or expression of a pair bond, either directly or indirectly , by their male partners. Introduction Preference for affiliative contact with a familiar individual is a fundamental part of social bonding in general, and of pair bonding in particular. Microtus ochrogaster (Wagner) (Prairie Voles) are well-known for displaying such affiliative bonds and thus have been used extensively in studies of the neurochemical, hormonal, and behavioral changes that underlie social bonding (Aragona and Wang 2004). Early studies focused on the presence or absence of mating as a critical factor in pair-bond formation (c.f., Carter et al. 1988), but those studies typically manipulated mating behavior of the animals via gonadectomy and/or hormonal treatments (c.f., Insel and Hulihan 1995). More recent studies, however, have suggested that natural variation in the normal hormonal and neurochemical processes that underlie Prairie Vole mating and social bonding can significantly affect the outcome of social-affiliation testing. This variation first came to light in a study of changes in the central nervous system associated with Prairie Vole pair bonding by Aragona et al. (Aragona et al. 2006). In that study, researchers included in their analysis only males from pairs in which the female was pregnant after 2 weeks of cohabitation, but did not take into account differences in pregnancy status (i.e., how far advanced the pregnancy was). Surprisingly, although the female mates exhibited some evidence of pregnancy after 2 weeks of cohabitation with a male, some of the males in those pairs did not show the neural reorganization necessary for pair-bond expression. Those 1Department of Pharmacology and Physiology, Oklahoma State University Center for Health Sciences, Tulsa OK 74107. *Corresponding author - tom.curtis@okstate.edu. Manuscript Editor: Joseph S. Johnson Northeastern Naturalist 542 K. McCracken, R. Lewis, and J.T. Curtis 2015 Vol. 22, No. 3 results suggest that the timing of the onset of his mate’s pregnancy may affect the formation, consolidation, or expression of a pair bond by the male. Results of other studies showed that both partner-preference expression and stranger-oriented aggression (social behaviors normally associated with pair-bonding) are heavily dependent on how soon after pairing female reproductive activation and pregnancy occur (Curtis 2010, Resendez et al. 2012). Mating behaviors of both members of a reproductive pair contribute to successful pregnancy (Coopersmith and Erskine 1994, Gray et al. 1974). Among rodents, the temporal patterns and numbers of mounts, intromissions, and ejaculations by the male can influence both egg fertilization, and the endocrine changes associated with pregnancy (Adler 1969). Female behavior also contributes to pregnancy success. Reproductive success (number of litters and pup survival) and offspring fitness were higher for females that chose the male with which they mated (Drickamer et al. 2000). Furthermore, reproductive success was enhanced when female rats controlled the frequency and temporal pattern of copulations (female-paced mating), as indicated by larger litter sizes (Coopersmith and Erskine 1994). The male Prairie Vole’s role in mating success is even more extensive than are those of male rats and mice. Female Prairie Voles do not display a spontaneous puberty. Rather, olfactory stimuli associated with the male stimulates a surge in circulating estrogen and induces female sexual receptivity, followed by copulation and ovulation (Carter et al. 1987). However, the latency to onset of sexual receptivity and mating can significantly affect subsequent male responses to his mate. Thus, factors that affect the latency to mating can have an inordinate impact on Prairie Vole pair bonding. There is evidence of female mate-choice in Prairie Voles (Pierce and Dewsbury 1991), but whether female pacing of mating in this species affects reproductive success and can influence male affiliative behavior is unknown. In the present study, we tested the hypothesis that female-paced mating can affect the affiliative behaviors of their male partners. We predicted that female pacing would reduce the latency to mating and/or other reproductive parameters, and thus change the males’ affiliative behaviors. We first examined the effects of female pacing on several reproductive parameters and then tested male behavior in a partner-preference paradigm. Materials and Methods All animal usage in this study was approved by the Oklahoma State University Center for Health Sciences Institutional Animal Care and Use Committee. The animals used in this study were adult (≥60 d old) male and female Prairie Voles from a captive breeding colony descended from a southern Illinois population (F4–F5 generation relative to the last out-crossing with wild stock). We maintained animals under a long-day photoperiod (14:10) and a colony-room temperature at 21 °C. Food (Purina® rabbit chow) and water were available ad libitum. From weaning at 19–20 days of age until used for experiments, we housed Prairie Voles as same-sex pairs in 10 cm x 17 cm x 28 cm cages with corn-cob bedding. We kept weaned males in a room separate from females and breeding pairs. Northeastern Naturalist Vol. 22, No. 3 K. McCracken, R. Lewis, and J.T. Curtis 2015 543 We assigned unrelated opposite-sex pairs of voles to one of 3 groups and placed them in an apparatus consisting of 2 parallel cages (10 cm x 17 cm x 28 cm) connected by a tube (7.5 cm in diameter x 16 cm in length). Both cages contained food and water sources. In the first group (n = 7 pairs), we tethered females to restrict their movements to 1 of the 2 cages, while the males were free to move at will between the occupied and empty cages. In the second group (n = 10 pairs), we tethered the males, and the females were free to move between cages. The third group (n = 9 pairs) consisted of pairs in which both animals were free to move about in the apparatus. We used low-light cameras to video-record all pairs for the first 72 h after pairing for later assessment of latency to mating and duration of mating period. We used a red light to provide sufficient illumination for video-recording during the lights-off period. In addition, we measured the animals’ locomotor activity during the first 24 h after pairing using customized computer software (R. Henderson, Florida State University, Tallahassee, FL) that monitored a series of light beams across the connecting tubes to record movements of the untethered animals between the cages. This program recorded the number of transits between cages and the amount of time spent by the subjects in each cage. After the initial 72-h video-recording period, we placed each pair in a clean 20 cm x 25 cm x 45 cm cage containing 1–2 cm of corn-cob bedding in the general colony room. We supplemented breeders with black-oil sunflower seeds and placed a ~10-cm-deep layer of Phleum pratense L. (Timothy) hay in their cages to provide environmental enrichment and nesting places. After a further 11 days of cohabitation, we tested males for evidence of pair-bonds with their partners. Partner-preference tests are routinely used to assess selective affiliation and examine pair-bonding in voles (Williams et al. 1992). The apparatus for our partnerpreference test consisted of a central cage (10 cm x 17 cm x 28 cm) joined by tubes (7.5 cm x 16 cm) to 2 identical parallel cages. One of the latter cages contained the familiar female partner with which the male had cohabited for 2 weeks and the other contained an unrelated, reproductively intact, sexually naïve, age-matched female with which the male had no prior contact. We tethered both females to restrict their movements to their separate cages, and thus, they had no direct contact with each other. All cages contained food and water. We released the subject male into the central cage where he had unfettered access to all 3 cages for 3h, and monitored his movements among the cages as described above. Throughout the test, we videorecorded the interactions of the animals for detailed behavioral analysis. Variables included the time spent by the male in each cage and the number of transits between cages (measures of general activity to ensure that treatments did not affect locomotor behavior) and the amount of time the male spent in quiet, direct contact with each female (a measure of affiliative behavior). We inferred a partner-preference if the males spent significantly more time in contact with their partners than they did with the strangers. Following behavioral testing, we euthanized females via CO2 asphyxiation and examined their uteri for evidence of pregnancy. We graded pregnancy status as described by Curtis (2010) using a 5-point scale that defines a gradient ranging Northeastern Naturalist 544 K. McCracken, R. Lewis, and J.T. Curtis 2015 Vol. 22, No. 3 from no evidence of reproductive activation to pregnancy sufficiently advanced to indicate that mating occurred within the first 48 hours after pairing. In addition, we noted the number of fetuses and their locations within the uterine horns. For pregnancies that were at very early stages, we noted the number and placement of implantation sites as indicated by dark red spots within the otherwise pink uteri. Finally, to assess whether female-pacing behavior could be related to differences in litter size associated with mating latency as has been noted for Microtus pennsylvanicus (Ord) (Meadow Voles) (Meek and Lee 1993), we retroactively examined data from 177 Prairie Vole pairs that were used in this and other studies. We compared litter size from pairs in which fetus size indicated early onset of mating, late onset of mating, and intermediate onset of mating relative to initial pairing. In general, we used Statistica software (Statsoft) to perform statistical analyses. We tested assumptions using Lavenes’s test for homogeneity of variance and half-normal plots for normality. We evaluated between-group treatment effects on physiological or behavioral measures using one- or two-way ANOVAs with sex and tethering status as factors. We employed repeated-measures ANOVA when measures were not independent (e.g., time split between multiple cages). We further examined significant (P < 0.05) main effects or interactions by conducting Student- Neuman-Keuls pair-wise comparisons and the Fisher-Freeman-Halton probability test (http://vassarstats.net/fisher2x3.html) to make group comparisons of mating parameters. Data are presented as mean ± SE. Results Demographics and behavior during cohabitation Animals in the 3 groups did not differ in age at the start of the experiment (range of group means ± SD = 87 ± 5 to 100 ± 11 d; all P-values for pair-wise comparisons ≥ 0.36). Tethering one of the animals affected the amount of time its partner spent in each of the cages during the initial 72 h of cohabitation (significant tethering x cage interaction: F2,17 = 5.98, P< 0.02; Fig. 1A). When females were tethered, their male partners spent an average of 55.6 ± 2.7 h of the 72-h cohabitation period in the cage containing the female and only 15.0 ± 2.8 h in the empty cage. These values did not differ from those of females when their male partners were tethered (57.5 ± 1.4 h and 13.0 ± 1.3 h, respectively). When neither animal was tethered, neither cage was favored. The average number of crossings between cages for males (688 ± 128) and females (799 ± 103) when their respective partners were tethered were not different. We excluded as an outlier locomotor-activity data from 1 male (>4 standard deviations above the group mean). Although there were no statistically significant effects whether or not that male was included, exclusion of that animal allowed a more accurate portrayal of locomotor behavior during the initial 72 h of cohabitation (Fig. 1B). The average number of cage crossings for the untethered pairs was somewhat higher (1028 ± 311) than for pairs in which 1 animal was tethered; however, the differences were not significantly different. Two males slipped out of their tethers but both did so shortly before (3 and 5 h, respectively) the end of the 72-h cohabitation period. Mating by these pairs had already ended; thus, we Northeastern Naturalist Vol. 22, No. 3 K. McCracken, R. Lewis, and J.T. Curtis 2015 545 did not consider this small amount of time without restriction sufficient to warrant excluding data for these individuals from the study. Female pacing and mating behavior or success Three pairs (2 pairs in which the male was tethered and 1 pair in which neither animal was tethered) did not mate during the initial 72-h cohabitation period. Examination of the females from these 3 pairs at the end of the 2-week cohabitation revealed 1 that displayed no evidence of reproductive activation, and 2 that displayed evidence of mating within the last 2–3 days of the 2-week cohabitation period. Several other pairs mounded bedding such that we could not fully assess some details of their behaviors, but we scored all of these pairs as maximally pregnant based on uterus examinations conducted at the end of the experiment. We observed mating during the first 72 h of cohabitation in all other pairs. The overall latency to first mating (39.7 ± 5.5 hours) was quite similar to the 39.5 ± Figure 1. Behaviors exhibited by newly paired Prairie Voles with various restrictions on freedom of movement among cages. (A) Amount of time spent in each half of a 2-cage apparatus by the male when the female was tethered to restrict her movements to a single cage, by the female when the male’s movements were restricted, and when neither animals’ movements were restricted. *indicates significantly less time spent in the unoccupied cage than in the cage were the partner was tethered (P < 0.001); ns indicates no significant group differences. (B) Number of cage crossings by males with tethered female mates, by females with tethered male mates, and by pairs in which neither animal was tethered. Northeastern Naturalist 546 K. McCracken, R. Lewis, and J.T. Curtis 2015 Vol. 22, No. 3 1.3 h reported by Curtis (2010) for reproductively intact female Prairie Voles, and there were no significant differences among the 3 tethering groups (F2,18 = 0.62, P = 0.55). The duration of mating (19.3 ± 2.4 h) also was similar to Curtis’ (2010) previous report (16.6 ± 2.5 h) and there were no group differences (F2,24 = 0.62, P = 0.27). Further, there were no differences in the numbers of mating bouts among pairs that mated. Female pacing did not alter any of the measures of mating success. When the male was tethered (i.e., mating was female-paced), 8 of 10 pairs mated successfully; when the female was tethered, 7 of 7 pairs mated successfully; and 8 of 9 pairs mated successfully when neither animal was tethered. These ratios were not statistically different (PA = PB = 0.76). Sixteen of 26 pairs achieved pregnancy within ~48 h, 7 pairs within ~96 hours, and only 3 pairs showed no signs of sexual activation earlier than about 10–11 days after pairing, if at all. In the present study, females that controlled the pace of mating carried a mean of 3.5 ± 0.7 fetuses at time of sacrifice. This value was not different from those for females that were tethered (4.1 ± 0.6 fetuses) or for females when neither animal was tethered (3.6 ± 0.6 fetuses). Examination of litter size (n = 177 litters) as a function of the timing of mating onset revealed no effect of mating onset (early mating onset = 4.15 ± 0.10 pups, intermediate onset = 4.31 ± 0.23 pups, and late = 4.71 ± 0.32 pups). Female pacing and male partner-preference performance Overall, measures of non-social behaviors during the partner-preference test did not differ between males in each of the 3 treatment groups. Locomotor activity, as measured by the number of crossings between cages, did not differ between groups (Fig. 2A). The patterns of cage occupancy (Fig. 2B) also did not differ among the 3 tethering groups. Regardless of group, males spent significantly more time during the test in their partner’s cage than in either the stranger’s cage or the empty cage (F2,48 = 41.74, P < 0.001; P < 0.02 for all pair-wise comparisons). Time spent in the empty cage and the cage in which the stranger was tethered did not differ. Males from all 3 groups spent significantly more time in quiet direct contact with their respective mates than they did with the strangers (F1,23 = 326.12, P < 0.001; P < 0.001 for all pair-wise comparisons, effect size = 0.93; Fig. 3), but the amounts of contact with the respective partners did not differ between groups (F2,23 = 0.23, P = 0.80; P > 0.43 for all pair-wise comparisons ). Discussion Pair-bonding by male Prairie Voles is strongly influenced by reproductive compatibility with their mates. Males display stronger partner preferences and higher levels of stranger-oriented aggression if their mates become pregnant within about 48–72 h after pairing than do males with partners that do not become pregnant or whose pregnancies are delayed (Curtis 2010, Resendez et al. 2012). Importantly, this behavioral variation appears to be tied to successful pregnancy rather than to the timing of copulation per se. Males that are paired with estrogen-primed, ovarectomized (and thus sexually receptive but incapable of pregnancy) females do not express partner Northeastern Naturalist Vol. 22, No. 3 K. McCracken, R. Lewis, and J.T. Curtis 2015 547 preferences despite copulating within the appropriate timeframe (Curtis 2010). Thus, other factors that affect reproductive success need to be explored. Previous studies examining vole-mating patterns generally have allowed both animals of a pair to move freely about the enclosure. The inability of a female to completely isolate herself from the male is a conspicuous limitation of these studies. For example, Corona et al. (2011) concluded that male behavior contributed to the pace of mating in Meadow Voles; however, in that experiment, the female was not given the option of separating herself from the male entirely. Although female Prairie Voles often reject the advances of males (J.T. Curtis, pers. observ.), this behavior does not afford the same magnitude of control by the female as when she can freely approach and withdraw from a restrained male. In the present study, we variously manipulated freedom of access of each member of mixed-sex pairs of Prairie Voles to their respective mates. In 1 group, the males’ movements were restricted, thus permitting the females to set the pace of mating, i.e., female pacing. In this Figure 2. Behaviors exhibited by male Prairie Voles during partner-preference testing. (A) Locomotor activity by males whose mates were tethered during the initial 72 h of a 2-week cohabitation, by males who were tethered during the early part of the cohabitation, and by males in which neither the male nor his mate were tethered during cohabitation. (B) Amount of time spent in each part of a 3-cage apparatus by males in each of the 3 groups described in (A). *indicates significantly less time spent in the empty cage or the stranger ’s cage than in the cage containing the familiar partner (P < 0.02). Northeastern Naturalist 548 K. McCracken, R. Lewis, and J.T. Curtis 2015 Vol. 22, No. 3 group, the females controlled the temporal pattern of social interactions with the male, the onset of mating, and the frequency of mating behaviors such as mounts, intromissions and, ultimately, ejaculations by the males. It might be expected that the females in this group would experience a lower level of stress relative to those in the other groups. In addition to the female-paced group, there were groups in which the females had less control over the pace of mating and thus, potentially experienced higher stress levels. Females in the group where neither animal was tethered may have had some control of mating because they could move away from the males. The third group consisted of pairs in which the females were tethered and, thus, had the least amount of control over interactions with the male, which in turn may have produced the highest stress levels. Stress interferes with pair-bond formation in female voles (DeVries et al. 1996); thus, non-paced mating could indirectly affect male behavior by altering female responses during partner-preference testing. Further, in rats, female-paced mating is rewarding (Jenkins and Becker 2003). If a similar response occurs in Prairie Voles, females may find paced mating less stressful than if the pace of mating is uncontrollable, again impacting female responses to the male during behavioral testing. However, our results indicate no such effects for Prairie Voles. Although female behaviors associated with mating do not appear to alter subsequent male behavior directly, the possibility remains that female pacing may affect reproductive outcomes, and thus affect male behavior indirectly. However, we did not detect this effect on male behavior. Neither the indices of non-social behavior nor the measures of reproductive success differed among the 3 treatment groups. Even when the male was tethered and the female had complete control of social interaction, the amount of time she spent in the cage containing the male did not differ from that occurring when the female was tethered and the male was in Figure 3. Social contact exhibited by male Prairie Voles during a partner-preference test. *asterisk indicates significantly more time spent in close, quiet contact with the familiar partner than with the stranger (P < 0.02). Northeastern Naturalist Vol. 22, No. 3 K. McCracken, R. Lewis, and J.T. Curtis 2015 549 control of the interactions. Similarly, female movements among the cages did not differ from those of males. Female pacing did not affect the proportion of pairs that mated, and did not affect latency to onset of mating or duration of mating. In fact, the latter 2 measures were remarkably similar to those previously reported for this species (Curtis 2010). Finally, our hypothesis that female-paced mating could affect subsequent male behavior by altering fertility was not supported because there were no group differences in litter size, nor was there a difference in litter size as a function of latency to pregnancy onset. A few caveats must be mentioned. We did not attempt to manipulate the timing, duration, number of intromissions, or the number of mating bouts. Thus, the present study differs from earlier similar mating studies that, for example, limited mating to 1 copulatory sequence (Coopersmith and Erskine 1994). The females in the present study were nulliparous, and it is unclear how parity (Dewsbury et al. 1979) may interact with female pacing in voles. However, at the time of parturition, female Prairie Voles likely have already formed social bonds and, thus, reduced mating success in post-partum pairs might not affect their subsequent social behavior. This phenomenon is an aspect of social behavior that has not been examined in Prairie Voles, and further study may provide insights into the stability of Prairie Vole pair-bonds. Finally, the present study examined the effects of female pacing on male behavior. This begs the obvious question: Why not test females? The answer is found in the study by Resendez et al. (2012) which showed that the effects of pregnancy status on pair-bond expression are sexually dimorphic in Prairie Voles. In that study, stranger-oriented aggressive behavior by males was positively correlated with their mates’ pregnancy status; however, they found no relationship between pregnancy status and aggression in females (Resendez et al. 2012). Thus, it appears that differential timing of pregnancy onset does not affect pair-bond formation in females as it does in males, so we focused our efforts on examining male behavior. Overall, the results of this study suggest that female paced-mating behavior does not directly or indirectly affect the behavior of her mate during subsequent partner-preference testing. Curtis (2010) suggested that non-volatile pheromonal signals from the female that carry information regarding the status of her pregnancy strongly influence male-preference behavior. We found nothing in the results of the present study to refute that suggestion. 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