Abstract
Male and female social presence has been known to induce/accelerate and suppress/retard mammalian estrous cycles under certain conditions. To further test this question, we placed nine female Siberian Hamsters (Phodopus Sungorus) hamsters with other female hamsters, nine with males, and seven alone. We measured their estrous stages with daily vaginal swabs over a period of eight days. Significantly more male exposed hamsters cycled than isolated hamsters, and close to significantly more female exposed hamsters cycled than isolated hamsters. These results suggest that social presence is related to increased cycling in Siberian hamsters.
Introduction
Most smaller non-primate mammals undergo estrous cycles of sexual receptiveness, which are characterized by changes in the lining of the genital tract and by changes in sexual aggressiveness and behavior (Gordon, 1977). In rodents, estrous cycles are usually four to five days long, and are represented by four stages (Gordon, 1977). Day 1 is estrous (indicated by the presence of cornified epithelial cells in the genital tract); Day 2 is metestrous (cornified epithelial cells and leukocytes); Day 3 is diestrous (leukocytes and nucleated epithelial cells); Day 4 is proestrous (nucleated epithelial cells) (Gordon, 1977; Zoology 468 Laboratory Manual). Longer cycles spend more days in diestrous than do shorter cycles.
Social presence has been shown to have acceleration/retardation and suppression/induction effects on mammalian estrous cycles (McClintock, 1983). Bronson (1974) suggested that the presence of female mice suppresses estrous in other female mice, and the presence of males induce or accelerate cycling in female mice. Marshlewska Koj et al. (1994) showed that high density crowding suppressed estrous cycling in mice. Erb et al. (1993) showed that presence of a male accelerated behavioral estrous by a day in Phodopus campbelli. It makes sense that females would expend energy to become sexually receptive in the presence of males, but not in the presence of females or alone.
In this experiment, we examined the effect of male presence and female presence on estrous cycling Siberian hamsters (Phodopus sungorus). We exposed nine female hamsters to another female, nine to a male, and isolated another seven hamsters, and performed daily vaginal swabs to assess their stages of estrous. Given the research of Bronson (1974) and Erb et al., (1993) we expected male social presence to induce or accelerate estrous cycling and female presence and isolation to suppress or retard estrous cycling.
Methods
Background
We used twenty-five adult (born between 8/12/98 and 8/25/98) sexually inexperienced female Siberian hamsters (Phodopus sungorus) as subjects and eighteen sexually experienced hamsters (nine females and nine males) as treatments. The subject hamsters were the first generation of offspring from the sexually experienced treatment hamsters, bred in the Swarthmore College Biology Department Colony, and kept in long-day cycles (LD 16:8). The subject hamsters were kept in same-sex cages of two or three and the treatment hamsters in cages alone. We provided the hamsters with water and Formulab 5008 (PMI International) food in excess throughout the experiment.
Pretreatment
On the afternoon of 11/16/98, we placed each of the subject hamsters into the larger half of a cage which was divided by wire mesh. We put pine shavings bedding, food, and water on both sides of every cage. We divided the hamsters into two groups of nine (male exposure experimental group and female exposure experimental group) and one of seven (control group), distributing litter mates evenly among the groups, and checking that mass was equally distributed between groups .
We placed the female exposure group with the control group in one sealed photochamber, and the male exposure group in another sealed photochamber, thus minimizing how much the male exposure hamsters were exposed to other females. Both photochambers remained on long day cycles (LD 16:8).
We left these hamsters alone for a week long equilibrating period. This week allowed each hamster to undergo at least one cycle in solitude and offset effects of their previous communal living before introducing any additional variables.
Treatment
On 11/23/98, we placed a sexually experienced male hamster in the empty half of each of the male exposure group cages, and a sexually experienced female hamster into the empty half of each of the female exposure group cages. We then allowed a second week-long equilibrating period, in order to allow any gradual effect on estrous cycles to appear.
We performed daily vaginal swabs on each of the subject hamsters between 4 and 5:30pm from 11/30/98 through 12/7/98. We dipped a micro-swab into saline solution, inserted it halfway into the hamster vagina, rotated it to obtain a sample of epithelial cells, and smeared the cells onto a slide which we later stained with Giemsa. We tried to avoid inserting the micro-swab so far as to touch the cervix and initiate a pseudo-pregnancy (Zoology 489 Laboratory Manual, 1993; Farel et al., 1996). We obtained both the microswabs and Giemsa stain from Fisher Scientific Company.
Analysis
We analyzed the swabs according to the percentages of cornified epithelial cells, nucleated epithelial cells, and leukocytes that we could identify in each sample (Zoology 489 Laboratory Manual, 1993). We deemed a hamster to be cycling if it showed estrous (exclusively cornified epithelial cells) twice within a three to seven day period.
We conducted two c2 contingency tests: one to determine whether the number of hamsters cycling in the control female hamsters differed from that in the male exposure group, and another to determine if it differed from that in the female exposure group.
Results
We found that significantly more of the hamsters exposed to male presence were cycling than isolated hamsters (N1=9, N2=7, c2=19.6, P=0.0118) (See Figure 2).
We were unsure as to the status of one of the female exposed hamsters: she was in estrous for two consecutive days, and in metestrous-to-diestrous for most of the other days. If we designated that hamster as cycling, significantly more hamsters exposed to female presence were cycling than isolated hamsters (N1=9, N2=7, c2=19.6, P=0.0118). (See Figure 2). If we designated that hamster as non-cycling, the difference between the number of cycling female exposed hamsters and cycling isolated hamsters was not significant, but close to significant (N1=9, N2=7, c2=14.4, P=0.0714).
We did not find any significant difference between the number of cycling male exposed hamsters and the number of cycling female exposed hamsters (counting questionable hamster as cycling: N1=9, N2=9, c2=0.0477, P=1; counting questionable hamster as not cycling: N1=9, N2=9, c2=7.54, P=0.4795).
We found one male treatment hamster dead in the cage on 12/1/98, with an excess of mucus at the end of its water bottle. The circumstances of its death resemble other similar deaths in the colony, so we believe it may be a genetic defect in the colony. We replaced it with another live sexually experienced male hamster.
Our data suggest that the presence of another hamster - male, and probably also female - is related to increased cyclicity in female Siberian hamsters. Our data also suggest that physical contact is not necessary for social presence to affect cyclicity; our subject hamsters were not in physical contact with their treatment hamsters.
These data make sense in light of Erb et al. (1993), who showed that the presence of males induce four day behavioral estrous cycles in Phodopus campbelli. Bronson (1974) also suggested that the presence of a priming factor in male laboratory mouse urine accelerates or induces estrous cycling. But Bronson (1974) and Champlin (1971) also suggested that the presence of females without male odor suppresses estrous cycling, which does not correspond with our data.
This difference may be due to species differences between hamsters and mice. It may also be due to our small sample size. A larger sample size would not only provide a more representative examination of cycling in female and male exposed hamsters compared to isolated hamsters, but would also allow a comparison between the effect of male hamsters and that of female hamsters. Hamsters might not have developed a pheromonal system that is as sensitive as that of laboratory mice, so that the presence of any hamster‹not just pheromones released by male hamsters‹stimulates sexual receptiveness. It would make sense to compare the effects of visual, olfactory and actual presence on cycling.
In any future experiments, I would recommend avoiding vaginal swabbing as a method of data collection as much as possible. Vaginal swabbing may indeed be the only way to collect reliable data on estrous cycling from rodents, but the process itself often seems cruel and unusual. The hamsters squealed and fight violently against being spread-eagled by a grip on neck fascia, and often squealed further while being swabbed.
AcknowledgmentsI'd like to thank my laboratory mates, Ilmi Granoff, Tirian Mink, Alex Robinson and Benson Wilder, for swabbing daily, sliding until all hours of the night, putting together great statistics, and giggling. Thanks to Sara Hiebert for ideas and assistance throughout the project, and the necessary push at the end: "So is this one cycling?" Thanks to Jocelyne Noveral for endless amounts of patience with the stories of suffering hamsters. Thanks to Susan Hunt, the most amazing bio WA on campus, for copious, directed and helpful comments. Thanks to John Kelly for generous supply of mesh and wire-cutters. Thanks to the Biology 20 classmates who shared the lab, the hamster-room-key, the cart, the scale, and the hamsters with us. And finally, thank you to the twenty-five subject hamsters (and the treatments, too, but not as much): we know it hurt, and we're sorry.
Literature Cited