- Email: [email protected]
Male-female interaction throughout the oestrous cycle of the Syrian hamster (Mesocricetus auratus)
Anim. Behav ., 1979,27, 91 9-929
MALE-FEMALE INTERACTION THROUGHOUT THE OESTROUS CYCLE OF THE SYRIAN HAMSTER (MESOCRICETUS AURA TUS) BY
ELIZABETH STEEL
MRC Unit on the Development and Integration of Behaviour, University of Cambridge Sub-Department of Animal Behaviour, Madingley, Cambridge CB3 8AA
Abstract. Interactions between male and female hamsters over the oestrous cycle were measured in terms of proximity . Independent variations in approaching and leaving by the female were found over the four-day period . Changes in female behaviour, which could be related to known changes in her hormonal state, resulted in changes in the male-female interaction not only at oestrous but also within the three dioestrous days of the cycle . These changes were in the amount of time the animals spent together or apart ; this in turn was affected by which sex initiated the approaches and leaves . During oestrus proximity measures correlated well with latency to first lordosis which is a sensitive measure of degree of receptivity . Proximity measures provide a detailed description of the male-female interaction and should prove useful in studying the effects of experimental alteration of previous experience and hormonal state . Two recent studies have provided detailed descriptions of the sexual behaviour of the Syrian hamster . Changes in female proceptive and receptive behaviour throughout an unmated oestrous period have been described by Beach et al . (1976) ; a study of male copulation has used the standard measures of the rodent copulatory series (Bunnell et al . 1977) . Although information is available on aggressive interactions (Payne & Swanson 1970) and on marking behaviour (Johnston 1977) there is no detailed study of male-female interaction over the entire oestrous cycle . The present study has three aims . First, to find a way to describe and to quantify malefemale interaction throughout the oestrous cycle using measures which are equally applicable to each day of the cycle . Second, to relate these measures to female receptivity during oestrus . Third, to relate changes in the male-female interaction to the hormonal state of the female throughout the four-day cycle . In a normal mating female hamsters appear, at first sight, to contribute little to the sexual interaction as they readily assume the lordosis posture and maintain it for long periods both between bouts of male mounting, and even during the post-ejaculatory interval . Experienced males rapidly elicit lordosis by chasing the female and licking her genital region . For this reason it is necessary to provide a situation where the female is free to show her interest in the male without lordosis being immediately elicited. In the present study an observation area consisting of two concentric runways 919
separated by coarse wire mesh was found appropriate . This allowed the animals to move freely and extensively while being able to approach and avoid each other at will . The behaviour was recorded both in terms of proximity (approaches and leaves by both sexes) and of overt sexual behaviour (female lordosis) . Methods Subjects were intact, sexually mature, multiparous females over three months old . They had been exposed to males for a brief period each day for several weeks to record their oestrous cycles. Males were intact and sexually experienced . Photoperiod . All animals were maintained on 14L : IOD with lights off at 11 .00 hours . Testing conditions. Tests before 11 .00 hours were made in white light ; those after 11 .00 hours in dim red light . Male-female pairs were tested in the apparatus shown in Fig . 1 . This consisted of two concentric runways separated by a wall of wire mesh . The hamsters were free to move independently in either direction and to sniff each other through the wire . A 10-min continuous record of the behaviour of both animals was made using a keyboard and WRATS event recorder (White 1971) . Test for lordosis with full access to male . After the 10-min runway test the male was transferred to a circular arena (72 cm diameter) . The female was then introduced to the same arena and watched for 2 min after the pair had made physical contact . The latency to lordosis (held for at least 3 s) was recorded.
9 20
ANIMAL BEHAVIOUR, 27,
Behavioural measures . The male was first put in the outer runway ; the female was then introduced into the inner runway and the recording started . The following measures were recorded 3 approaches 9 ; 9 approaches a ; d leaves Y ; leaves a; ? lordosis . Definition of Behavioural Measures Time together : recorded when the animals were less than 10 cm apart . Time apart : recorded when they were over 10 cm apart . Approach : distance between animals decreases from over 10 cm to less than 10 cm . Leave : distance increases from less than 10 cm to over 10 cm . Approachings and leavings were not recorded if the male and female ran past each other without pausing . Lordosis : this included a graded series of postures from an immobile crouched posture
Observer
A
B I Perspex
®1x2 cm . wire mesh
White pointed hordboord
Fig. 1 . Plan (above) and cross-section along the line A-B (below) . Animals were free to move in the concentric runways and to investigate each other through the wire partition. Clear Perspex walls allowed the observer at position B to see all interactions . Width of runways, 8 . 5 cm ; length of outside wall, 72 cm ; height of walls, 34 cm .
3
with the tail touching the floor to the full lordosis posture with the tail elevated above the general level of the back. Tests During the Oestrous Cycle Twelve animals were tested once on each of days 2, 3 and 4 between 14 .30 and 15 .30 hours. The tests spanned 4 oestrous cycles and were randomized so that the first test could be on any dioestrous day of the cycle . Tests were not necessarily on successive days of the cycle . On day 1 (oestrus) 11 of the 12 animals were tested four times, the tests being separated by 2 .5-h intervals. Testing session began at 09 .40, 12 .12, 14.44 and 17 .15 hours and lasted for 60 min, so that up to four animals could be tested on any one day . Analysis of Data From the measures recorded on the keyboard the following derived measures were obtained for analysis from each 10-min test : Total female approaches 1 . YAp % = Total approaches by male and female Total female leaves 2 . ?L% = 0 Total leaves by male and female . Female Index of Approach = YAp % minus 3 ?L This gives an overall index of female approach or avoidance . It is negative if the female leaves more than she approaches and positive if she approaches more than she leaves (Hinde & Atkinson 1970) . Measures of approaching and leaving were based on the time when both animals were free to interact . If a female showed lordosis in the runway test, the time available for free interaction was less than 10 min . All subsequent analysis is based on time when the female was not in lordosis, i .e . all male approaches and leaves made while the female was in lordosis are dropped from the record . Statistics Non-parametric statistical tests were used to assess the data (Siegel 1956) . Comparisons between the same animals on different days were made using the Wilcoxon matched-pairs test . * No predictions were made about the direction *Comparisons between independent sub-groups used the Mann-Whitney U test .
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
of the differences and two-tailed probabilities were used throughout . Spearman rank correlation coefficients (rs) were calculated to assess the relationship between various measures . The Kendall coefficient of concordance (W) was used to assess the degree of agreement among animals for various measures over the four tests during oestrus . Results The aim of the study was to compare the behaviour of oestrous females with their behaviour on other days of the cycle. For this purpose a single runway test from day 1 was required for each female, which best represented her full oestrous state . The first stage of the analysis seeks to relate measures of lordosis (receptivity) to the runway measures of approaching and leaving . Receptivity During Oestrus Lordosis in arena. See Table I . Arena tests with full access to a male revealed that all 11 females were in oestrus . Five showed lordosis on all four tests, four on three tests, and two on two tests. On test 3 (4 to 5 h after lights off ) all showed lordosis . The median latency to lordosis was shortest on test 3 . The Kendall coefficient of concordance (W) for the four tests was 0 . 30 (P < 0 .01) showing that there was a significant agreement between females on this latency measure . Lordosis in runway. See Table I . Only eight females showed lordosis in the runway, although all showed it on at least two tests in the arena . Of the eight females, five showed lordosis on only one test, one on two tests and two on three tests. Table I . Measures of Lordosis for 11 Oestrous Females Tested at 2 . 5-h Intervals both in the Runway and in the Arena with Full Access to a Male
921
Lordosis duration varied from 11 % of a test to 89 % ; the median longest duration for the eight females was 59 % . One female showed her longest duration on test 1, three on test 2, two on test 3 and two on test 4 . Relation between lordosis in runway and arena . Eight females showed lordosis in the runway ; their median arena latency immediately after they had showed runway lordosis was 3 . 8 s while their shortest arena latency after runway tests without lordosis was 13 . 0 s (Wilcoxon test, P < 0 . 05) . The shortest arena latency for females who showed runway lordosis was 2 . 5 s and for the three females who never showed runway lordosis it was 9 . 0 s (Mann-Whitney U test, P < 0.05) . Lordosis in the two situations correlates well . Animals who show runway lordosis are likely to show a very short latency to lordosis in the arena when tested immediately afterwards . Females who never showed runway lordosis have longer latencies (at best) in the arena than females who show runway lordosis . Relation between lordosis in the arena and SAp %, ?L% and female index. Figure 2 shows the medians of these four measures plotted for the four tests during oestrus . As already shown, females tend to show their lowest latency to lordosis on the same test . Similar Kendall coefficients of concordance (W) are not significant for the runway measures of ?Ap % (0 . 02), 9L% (0 . 14) or female index (0 . 17) ; that is, animals do not show their maximum scores for these measures on the same tests . It follows that
so
FEMALE INDEX - .20
60
0
SECONDS 120
Oestrous test 40
1
2
3
4
No . showing lordosis 6 Median latency (s) 120 No. showing their shortest latency 0
9 28
11 8
10 13
3
4
4
1
5
4
3
1
3
2
2
All tests
Lordosis in arena
20
11/11 0 z OFSTRUS TEST
Lordosis in runway
No. showing lordosis No. showing their maximum lordosis duration
8/11
Fig. 2. Four tests were conducted at 2 . 5-h intervals during oestrus starting at 09 .40 (80 min before lights-off), 12 .12, 14.44 and 17.15 hours. Four measures (medians, N = 11) are plotted for each of these tests . • : latency to lordosis in the arena ; 0, 9Ap% ; o, ?L% ; A, female index .
9 22
ANIMAL BEHAVIOUR, 27,
latency to lordosis in the arena correlates weakly with these three measures . Table II shows that the female index was the most highly correlated, showing a significant negative relationship to lordosis latency on three out of four tests . A comparison of the measures ~Ap %, ?L and female index for tests where females were most receptive and least receptive (measured by latency to lordosis in the arena) show that only female index differs significantly between the two conditions (Table III) . Female Ap differs by only 5 % ; ?L % differs by 16 %, but with a large variance . The net balance between approaching and leaving is thus the best single indicator of potential receptivity . Relation between lordosis in the runway and lAp %, ?L % and female index . For the eight females who showed lordosis in the runway, the median scores for ~Ap %, ?L % and female index were calculated for tests with and without lordosis (Table IV) . The female index was more positive (P < 0 . 05) and ?Ap % was greater (P < 0 . 05) on those tests where lordosis occurred ; ~L % were slightly higher on tests without lordosis but not significantly . Table V shows the most extreme scores for the three females who never showed lordosis and for the eight females who showed some lordosis in the runway. The females who showed lordosis
3
also attained a greater YAp % score (P < 0 .05) and a more positive female index (P < 0 . 05), than did females who never showed lordosis . Females showing lordosis tended to leave less but this difference was not significant . Discussion. The evidence reviewed so far indicates that receptivity increases throughout the day of oestrus and reaches a peak about 5 h after lights-off (cf. Beach et al . 1976) . Measures of approach and avoidance by the female show an overall relationship with measures of receptivity although their variation with the time of day is not nearly so marked . Lordosis in the arena and in the runway are correlated and both relate most consistently to the female index . As the female index becomes more positive, that is as the balance between female approach and female leaving shifts towards relatively more approach, so the females are likely to be more receptive . However, animals vary in the rate at which they attain full oestrous behaviour and tests at each time of day contain animals at different stages of oestrus . As the female index was correlated with latency to lordosis in the arena and was related to the occurrence of lordosis in the runway, the oestrous test with the highest (most positive) female index for each animal was used to compare with the other three days of the oestrous cycle .
Table II . Spearman Rank Correlation Coefficients (rs) between the Latency to Lordosis in the Arena and Three Measures of Female Behaviour in the Runway during the Four Oestrous Tests Oestrous tests Correlation of lordosis latency with ?Ap %
Female index
1
2
- 0 . 29
0 . 18
+0-71 P < 0 . 05 -0-66 P < 0 . 05
-0 . 16 - 0 .23
3
4
- 0 .70 P < 0 .05 -0 .23
-0-42
- 0 . 53 P < 0.1
-0-59 P < 0.1
+0 . 32
Table III. This Shows the Corresponding Median YAp %, ~L % and Female Index for Tests Preceding Each Animal's Highest and Lowest Latency to Lordosis in the Arena Lordosis latency (s)
?Ap %
~L %
Female index
Test with : Lowest latency
6
38
58
- l
Highest latency
120
33
74
- 27
NS
NS
Wilcoxon test
P < 0 .05
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
923
Table IV. Comparison of YAp %, YL% and Female Index between Tests with and without Lordosis for Eight Oestrous Females Median for tests
YAp
YL%
With lordosis
60
57 . 5
+ 19 . 5
Without lordosis
30 .5
65
- 25
P < 0 . 05
NS
P < 0 .05
Wilcoxon test
Female index
Table V. From Four Tests during Day 1, Tests with the Highest ?Ap %, the Lowest 2L and the Most Positive Female Index, for the Females who never Showed Lordosis and those who Showed Lordosis
N Females never showing lordosis Females showing lordosis
Highest ?Ap %
Lowest ?L %
Most positive female index
3
35
66
- 33
8
74 .5
33
+ 23 . 5
NS
P<0 . 05
Mann-Whitney U test
P<0 .05
Tests during dioestrus were conducted 3 . 5 to 4 . 5 h after lights-off, corresponding to the period of maximum receptivity on day 1 . Behavioural Changes over the Oestrous Cycle The next part of the analysis looks at changes over the oestrous cycle . The first section concentrates on changes in the measures of approaching and leaving already described and the relationship between them . In the later sections new measures (derived from the approachleave data) are introduced and described . Relationship between approaches and leaves . Figure 3 shows the group median scores for three measures on each day of the oestrous cycle . The female index, which reflects the balance between approaching and leaving, rises significantly between days 3 and 4 and over dioestrus (days 2 to 4) ; it shows a further marked rise at oestrus . The percentage of female leaves (~L %) falls significantly between days 3 and 4 and over dioestrus ; this trend is continued into oestrus . The percentage of female approaches (SAp %) rises significantly over dioestrus but most between days 2 and 3 ; again there is a marked rise at oestrus . Changes in female index could reflect changes in the proportion of approaches and/or of leaves . It is clear from the graph that both measures change over the 4-day period . The female index correlates strongly with both measures, having an inverse relation-
DAYS 2-4 0 4A % p<0O1 o 9L% p< 0. 001 4 91NDEX P<0.001 9 INDEX *20
I 100
80
0
60
-20
40
-40
20
t
3
4
1
-60 1 0
2
-80
DAY OF OESTROUS CYCLE
Fig . 3 . The medians for YAp %, YL % and Y index are plotted for each day of the oestrous cycle. The same data for day 2 is plotted twice . The significance of differences between adjacent days are shown on the graph : *P < 0 . 05, **P < 0 . 01, ***P < 0 . 001 . The significance of changes over dioestrus (day 2 - day 4) are also shown .
ANIMAL BEHAVIOUR, 27, 3
9 24
ship with YL Y. and a positive relationship with ?Ap % on each of the four days (Table VI) . The different shapes of the two curves representing ?Ap % and ?L % suggest that these two measures are not varying together . This is borne out by the correlations between them which are low and non-significant on each day . Probabilities of approaching and leaving. In a sequence of approaches and leaves by two interacting animals each of the four events male approach, female approach, male leave and female leave, must be preceded or followed by one of two possible events . For example, after a male approach there must follow either a male leave or a female leave . Similarly, before each male approach there must be either a male leave or a female leave . The percentages of male and female moves preceding and following each of the four events named above were calculated for each day of the cycle and are displayed in Fig. 4. These percentages can be interpreted as probabilities of approaching and leaving and will be referred to as probabilities throughout the rest of the paper. Bouts of time together and time apart . The complete 10-min sequence of male-female interaction consists of alternating bouts of time spent together and time spent apart . Each bout of time together (or apart) may be initiated by either sex and ended by either sex . Thus there are eight different possible types of bout in the sequence . In Figs . 5 and 6 are plotted the four types of bout accounting for time spent together and the four types of bout accounting for time spent apart. These show the total time spent in each type of bout as a percentage of total test time .
Each type of bout accounts for a different proportion of the observation time . The time that any one of these eight types of bout contributes to the total test time may be affected by the number of such bouts and/or by their duration . For example, if one type of bout accounts for 30% of the total time, it may be because there are numerous short bouts of this particular type or because there are fewer but longer ones. Figures 5 and 6 show that the two graphs depicting the number of bouts and their total time follow each other closely in most cases . However, both the number and occurrence of these bouts depends on how readily the male and female approach and leave one another. To estimate this parameter mean bout lengths were calculated for each type on each day of the cycle . The shortest bouts were YL-SAp (group mean on day 2 = 4 . 8 s) and the longest were SAp- Sl, (group mean on day 2 17 . 6 s) but there was considerable variation both between animals and between days . In order to assess the changes between days the mean score for each animal was calculated as a percentage deviation from that animal's mean score for all four days of the cycle . Table VII shows whether bouts became longer or shorter over dioestrus, between days 4 and oestrus, or over the whole cycle. It can be seen that the durations of four types of bout change significantly at some stage of the cycle and that changes in one direction over dioestrus are frequently reversed at oestrus . Discussion . The previous analysis has shown that approaching and leaving are not correlated and can vary independently. The percentage of female approaches and the percentage of female
Table VI. Spearman Rank Correlation Coefficients (r,) between YAp %, 9L % and the Female Index for Each Day of the Oestrous Cycle Day of oestrous cycle 2
3
4
1
Y Index : ?Ap
+0-60 P < 0 .05
+0. 62 P<0 . 05
+0 . 64 P<0 . 05
+0 . 84 P<0.001
? Index : YL%
-0-57 P < 0 .05
-0-67 P<0 . 05
-0-77 P<0 . 01
-0-49
+0-28
-0-13
-0-04
NS
NS
NS
rs
correlation between
9Ap% : ?L%
N= 12
NS
0 . 00 NS
N= 11
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
leaves change significantly over the oestrous cycle, but they change at different rates . Changes in the probability of one event (an approach or a leave) occurring with increasing time since the previous event must underlie the other behavioural changes observed in this analysis . The overall probability of a particular bout type ending with time since its initiation, is crudely reflected in its mean length . Changes in bout lengths must affect the number and total duration of bouts of time spent together and apart (Figs . 5 and 6) . Concurrent changes over the cycle in the probability of male approaches (or leaves) and female approaches (or leaves) After a male approach
After a female approach
925
after a specific event will affect the overall probabilities of approaching and leaving shown in Fig. 4 . Mean bout lengths vary widely and it is likely that the length of each bout is affected not only by the immediately preceding leave or approach but by a whole range of events that have occurred previously during the test . Significant changes in probabilities of approaching and leaving (Fig . 4) and in bouts of time spent together and apart (Figs . 5 and 6) fall into three categories . Some changes occur only during dioestrus (days 2 to 4), some occur between day 4 and day 1 (oestrus), while some occur during dioestrus and again at oestrus . After a mal¢ Icovo
After a female leave
1/. dA
°/o
O* A
A N S c/o I
Before a male approach
Before a female approach
+l» Cr L
Before a male leave
+/o
d' A
»/o
A
Before a female leava
250-
2
3
4
1
2
2
3 4 1 2 2 3 4 1 DAY OF OESTROUS CYCLE
2
Fig . 4 . The four graphs on the left show the percentage of female leaves and the percentage of male leaves which either follow or precede all male approaches and all female approaches ; the four graphs on the right show the percentage of female approaches and the percentage of male approaches which either follow or precede all male leaves and all female leaves . Group medians are given for each day of the oestrous cycle . The same data for day 2 is plotted twice. L = leaves ; A = approaches . The following significant differences are shown by asterisks : (1) Asterisks above each graph show significant differences between male and female percentages on any single day, e.g. a male approach on day 2 is significantly more often followed by a female leave than by a male leave (top left-hand graph) . (2) Asterisks on the graph show significant changes in percentage between adjacent days of the cycle. (3) Asterisks on the bar at the bottom left of the graphs show significant changes in percentage between days 2 and 4 (i .e . over the three dioestrous days of the cycle) . *p < 0-05 ; **,P < 0 . 01 ; ***,P < 0 .001 ; Ns = not significant .
926
ANIMAL BEHAVIOUR, 27,
For most measures the extreme points on the graph are on day 1 and day 2 .
not show lordosis in the double runway . Even at the height of oestrus it was possible to collect data on her interaction with the male . Second, the analysis of the approach-leave data revealed very subtle changes in the male-female relationship which could be readily quantified . The lordosis quotient used to measure degrees of receptivity in the female rat is not applicable to hamsters as the female assumes lordosis before the male mounts . Hamster receptivity has been measured as total time spent in lordosis during a test of given length, as latency to the first lordosis, and as duration of the longest-held lordosis (Carter et al . 1973 ; Carter & Porges 1974) . My own observations (unpublished) have shown,that the latency to the adoption of the lordosis posture is dose dependent over a wide range of oestrogen doses (1 to 30 .tg oestradiol benzoate with 500 gg progesterone) . Latency to lordosis is thus likely to be a good measure of intensity of oestrus in the present study. How-
General Discussion The initial aim of this study was to find a way of describing and quantifying male-female interactions; it was hoped to use such a method to show changes in the male-female relationship between dioestrus and oestrus . By concentrating on proximity measures (bouts of time spent together and apart) rather than overt behaviour patterns, it was intended that the same measures could be collected on each day whether or not the females were in oestrus. The type of apparatus used was dictated by a need to prevent the oestrous female from spending most of the observation period in lordosis . This method has been successful in two ways . First, a female in the double runway assumes the lordosis posture less than when she is given free access to a male ; on many oestrous tests females, who were receptive in the arena, did OfA-9L
3
OA -dL
9A-9L
9A- d'L
40CF
CZ':
353025 20 151050-
1
2
3
4
1
i 2
I
2
3
I
1
4 1 2 2 3 4 DAY OF OESTROUS CYCLE
1
Fig . 5 . The two left-hand graphs refer to bouts of time spent together which were initiated by the male, and the two right-hand graphs to bouts of time spent together initiated by the female . Group medians are plotted for each day of the oestrous cycle. The same data for day 2 is plotted twice. --*--, time spent together as a percentage of total test time . --0--, number of bouts of time together as a percentage of total number of all bouts both together and apart . The following significant differences are shown by asterisks : (1) Asterisks on the graph show significant changes in percentage between adjacent days of the cycle . (2) Asterisks on the bars above each graph show significant changes in percentages between days 2 and 4 (i .e . over the three dioestrous days of the cycle) . White bar = % of total time ; black bar = of total number of bouts . Ns = not significant ; *P < 0 . 05 ; **P < 0 . 01 ; ***P < 0 . 001 .
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
ever, it must be remembered that immediately before the test used to measure lordosis latency, the male and female have been together for 10 min in the double runway . This latency measure also has the advantage that the male can be separated from the female before intromission CL-9A
takes place . This eliminates the risk of inducing pseudopregnancy, an important point if daily changes in the subsequent oestrous cycle are to be studied. Lordosis is broadly related to the female index which in turn is based on female approaching
Cf L-CTA
YL-CYA
40c .€7
927
YL-OA
F1.4Y
C€SJ
O
35300
25-
1
201510-
0
5 O
-
~ i
2
c
o
o'
3
1
I
2
2
i
1
1
4 1 2 2 3 4 DAY OF OESTROUS CYCLE
1
2
3
4
Fig . 6 . The two left-hand graphs refer to bouts of time spent apart which were initiated by the male and the two right-hand graphs to bouts of time spent apart initiated by the female . Group medians are plotted for each day of the oestrous cycle . The same data for day 2 is plotted twice . -h, time spent apart as percentage of total test time . --0--, number of bouts of time apart as a percentage of total number of all bouts both together and apart . Significant differences are shown as in Fig . 5 . Table VII . Changes in Bout Lengths between Days of the Oestrous Cycle. Bout Lengths for Each Day Were Expressed as a Percentage Deviation from the Mean Bout Length Over all Four Days . This Table Shows the Difference in this Percentage Deviation Between Days of the Cycle and Indicates whether Mean Bout Lengths Increased by over 10 % (+), Decreased by over 10 % (-) or Changed by Less than 10 % (0) Days 2 to 4 change
Days 4 to 1 % change
Days 1 to 2 % change
Bouts together
dA-dL
-
YA-?L
-
A-dL
-
33 .4 45 . 2 P < 0 . 05 62 . 4 P<0 . 05 63 . 9 P < 0 . 05
+ +
37 . 4 45 .4
0 0
4.0 0.2
+
65 . 4 P<0 .01 41 . 1
0
3 .0
+
22 . 8
9.2
+
9. 1 5.5 49 . 6
0 +
87 . 0 P<0 .01 6.3 38 . 3 16 . 9
Bouts apart
d L-9A
-
SL- o'A YL-9A YL-TA
+
0 96 .2 P < 0 . 001 0 2.8 32 .8 0 66 . 5 -
1
2
928
ANIMAL BEHAVIOUR, 27, 3
and leaving . The female index does not fluctuate in precise synchrony with changes in degree of receptivity, probably because it is affected more by subtle interaction with the male than is lordosis itself. The more quickly a female showed lordosis in the runway situation, the less data on male-female interaction were collected as female lordosis time had to be deducted from the 10-min test data . There is thus a sampling problem which makes it more difficult to collect good approach-leave data from females who are highly receptive . As expected, many changes in the malefemale interaction between oestrus and dioestrus were shown by this analysis ; however, the similarly large changes within the three days of dioestrus were not expected and merit some discussion. A second unexpected finding was that approaching and leaving were not correlated and thus varied independently . This leads to almost infinite variation in the fine details of the male-female interaction as approaching and leaving change at different rates over the cycle . The most obvious independent variable associated with the oestrous cycle is the hormonal state of the female . While this is known to change over the four days, there is no a priori reason to assume that the hormonal state of the male changes also . The observed changes in the male-female relationship can be related to known hormonal changes throughout the oestrous cycle . Baranczuk & Greenwald (1973) measured plasma oestradiol at intervals over the entire cycle . For the first 11 days oestradiol (at less than 20 pg/ml) is barely above the level in ovariectomized females . Towards the end of day 2, plasma levels begin to rise and reach about 90 pg/ml 24 h later . During day 4 there is a further rapid increase (up to 180 pg/ml) which initiates the ovulatory LH surge . After this oestradiol falls rapidly to its lowest level on day 1 ; behavioural oestrus begins after oestradiol starts to decline, and continues (in unmated females) for about the next 16 h while oestradiol is at its lowest . Progesterone levels rise (from about 7 ng/ml) through the second half of day 4 and peak (16 ng/ml) during the dark phase of day 1 while behavioural oestrus is at its height (Norman & Greenwald 1971) . In the present study females avoided the male most on day 2 when oestrogen levels are at their lowest ; my own unpublished observations show that high avoidance is characteristic of ovari-
ectomized untreated females and may thus be a consequence of low oestrogen levels . For a group of eight ovariectomized females tested in the double runway at the same time of day as dioestrous females in the present study, ?Ap was 17 %, 9L % was 79 % and the female index was - 64 .5. These figures are almost identical with those of day 2 females (Fig . 3) . Although female behaviour changes over days 2 and 3 most of the significant changes occur during days 3 to 4 when oestrogen levels are rising rapidly . Further changes occur during days 4 to 1 including the appearance of receptive behaviour. To what extent these behavioural changes can be separately attributed to oestrogen and progesterone is a matter for further study, though in preliminary experiments changes in female approaching and leaving have been obtained with oestrogen alone, as has lordosis, after several days of treatment (unpublished observations ; see also Carter et al . 1973) . Changes in the female's hormonal state could have far-reaching effects on the male-female relationship . First of all, hormone-dependent changes in the female's behaviour could affect the male ; consequent changes in his behaviour could in turn affect the female . Hormonedependent changes in the female which attract her to the male or which attract the male to her could also affect the behaviour of both partners. For example, it is known that olfactory cues from the female attract the male and increase his likelihood of mounting (Murphy 1973 ; Landauer et al. 1978). Both males and females produce ultrasonic calls . Females in oestrus call more than non-oestrous females and the rate of calling of both sexes is increased in the presence of the sexual partner (Floody & Pfaff 1977 ; Floody et al . 1977) . In conclusion, the present analysis is based on measures which were obtained in an artificial situation where the animals did not have free access to one another. Interpretation of the data should be guided always by observations made in a more natural interaction . One advantage of the apparatus used is that the animals can move freely and extensively and the artifacts of testing in a small arena are avoided. By eliminating the opportunity for the display of aggression and most lordosis (except at the height of oestrus) more subtle aspects of the female's behaviour can be revealed and recorded quantitatively . The second advantage is that analysis of a relationship in terms of approaches and leaves allows
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
as on the other days of the cycle as it does not rely on specific behaviour patterns which may be present on some days of the cycle and not on others . However, it does not preclude other relevant behaviour being recorded and superimposed on the sequence of proximity measures (Steel 1979, in press) . A subsequent paper shows the value of this method in exploring the roles of both partners in the complex and continually changing malefemale relationship during the oestrous cycle (Steel 1979, in press) . REFERENCES Baranczuk, R. & Greenwald, G. S . 1973 . Peripheral levels of oestrogen in the cyclic hamster . Endocrinology, 92, 805-812 . Beach, F . A ., Stern, B ., Carmichael, M. & Ranson, E. 1976. Comparisons of sexual receptivity and proceptivity in female hamsters. Behav . Biol., 18, 473-487 . Bunnell, B . N ., Boland, B . D . & Dewsbury, D. A . 1977 . Copulatory behavior of golden hamsters (Mesocricetus auratus). Behaviour, 61, 180-206 . Carter, C. S . & Porges, S. W. 1974. Ovarian hormones and the duration of sexual receptivity in the female golden hamster. Horm. Behav., 5, 303-315 . Carter, C. S ., Michael, S . J. & Morris, A . H. 1973 . Hormonal induction of female sexual behaviour in male and female hamsters . Horm . Behav ., 4, 129-141 . Floody, O . R . & Pfaff, D . W . 1977 . Communication among hamsters by high frequency acoustic signals. III. Responses evoked by natural and synthetic ultrasounds . J. comp . physiol. Psychol ., 91, 821-830.
929
Floody, O . R., Pfaff, D . W . & Lewis, C. D. 1977 . Communication among hamsters by high-frequency acoustic signals. 11 . Determinants of calling by females and males . J. comp. physiol. Psychol., 91, 807-820. Hinde, R . A . & Atkinson, S . 1970. Assessing the roles of social partners in maintaining mutual proximity, as exemplified by mother-infant relations in rhesus monkeys . Anim . Behav., 18, 169-176 . Johnston, R . E . 1977 . The causation of two scent marking behaviours in female hamsters (Mesocricetus auratus) . Anim . Behav., 25, 317-327 . Landauer, M. R ., Banks, E. M. & Carter, C . S . 1978 . Sexual and olfactory preferences of naive and experienced male hamsters . Anim . Behav ., 26, 611-621 . Murphy, M . R. 1973 . Effects of female hamster vaginal discharge on the behaviour of male hamsters . Behav . Biol., 9, 367-375 . Norman, R . L. & Greenwald, G . S . 1971 . Effect of phenobarbital, hypophysectomy and X-irradiation on preovulatory progesterone levels in the cyclic hamster . Endocrinology, 89, 598-605. Payne, A P . & Swanson, H . H . 1970. Agonistic behaviour between pairs of hamsters of the same and opposite sex in a neutral observation area. Behaviour, 36, 259-269 . Siegel, S . 1956 . Nonparametric Statistics for the Behavioral Sciences. New York : McGraw-Hill . Steel, E . 1979 . Changes in female attractivity and proceptivity throughout the oestrous cycle of the Syrian hamster (Mesocricetus auratus) . Anim . Behav., 27, in press. White, R. E . C . 1971 . WRATS : A computer compatible system for automatically recording and transcribing behavioural data . Behaviour, 40, 135-161 . (Received 23 October 1978 ; revised 6 December 1978 ; MS. number : 1824)
MALE-FEMALE INTERACTION THROUGHOUT THE OESTROUS CYCLE OF THE SYRIAN HAMSTER (MESOCRICETUS AURA TUS) BY
ELIZABETH STEEL
MRC Unit on the Development and Integration of Behaviour, University of Cambridge Sub-Department of Animal Behaviour, Madingley, Cambridge CB3 8AA
Abstract. Interactions between male and female hamsters over the oestrous cycle were measured in terms of proximity . Independent variations in approaching and leaving by the female were found over the four-day period . Changes in female behaviour, which could be related to known changes in her hormonal state, resulted in changes in the male-female interaction not only at oestrous but also within the three dioestrous days of the cycle . These changes were in the amount of time the animals spent together or apart ; this in turn was affected by which sex initiated the approaches and leaves . During oestrus proximity measures correlated well with latency to first lordosis which is a sensitive measure of degree of receptivity . Proximity measures provide a detailed description of the male-female interaction and should prove useful in studying the effects of experimental alteration of previous experience and hormonal state . Two recent studies have provided detailed descriptions of the sexual behaviour of the Syrian hamster . Changes in female proceptive and receptive behaviour throughout an unmated oestrous period have been described by Beach et al . (1976) ; a study of male copulation has used the standard measures of the rodent copulatory series (Bunnell et al . 1977) . Although information is available on aggressive interactions (Payne & Swanson 1970) and on marking behaviour (Johnston 1977) there is no detailed study of male-female interaction over the entire oestrous cycle . The present study has three aims . First, to find a way to describe and to quantify malefemale interaction throughout the oestrous cycle using measures which are equally applicable to each day of the cycle . Second, to relate these measures to female receptivity during oestrus . Third, to relate changes in the male-female interaction to the hormonal state of the female throughout the four-day cycle . In a normal mating female hamsters appear, at first sight, to contribute little to the sexual interaction as they readily assume the lordosis posture and maintain it for long periods both between bouts of male mounting, and even during the post-ejaculatory interval . Experienced males rapidly elicit lordosis by chasing the female and licking her genital region . For this reason it is necessary to provide a situation where the female is free to show her interest in the male without lordosis being immediately elicited. In the present study an observation area consisting of two concentric runways 919
separated by coarse wire mesh was found appropriate . This allowed the animals to move freely and extensively while being able to approach and avoid each other at will . The behaviour was recorded both in terms of proximity (approaches and leaves by both sexes) and of overt sexual behaviour (female lordosis) . Methods Subjects were intact, sexually mature, multiparous females over three months old . They had been exposed to males for a brief period each day for several weeks to record their oestrous cycles. Males were intact and sexually experienced . Photoperiod . All animals were maintained on 14L : IOD with lights off at 11 .00 hours . Testing conditions. Tests before 11 .00 hours were made in white light ; those after 11 .00 hours in dim red light . Male-female pairs were tested in the apparatus shown in Fig . 1 . This consisted of two concentric runways separated by a wall of wire mesh . The hamsters were free to move independently in either direction and to sniff each other through the wire . A 10-min continuous record of the behaviour of both animals was made using a keyboard and WRATS event recorder (White 1971) . Test for lordosis with full access to male . After the 10-min runway test the male was transferred to a circular arena (72 cm diameter) . The female was then introduced to the same arena and watched for 2 min after the pair had made physical contact . The latency to lordosis (held for at least 3 s) was recorded.
9 20
ANIMAL BEHAVIOUR, 27,
Behavioural measures . The male was first put in the outer runway ; the female was then introduced into the inner runway and the recording started . The following measures were recorded 3 approaches 9 ; 9 approaches a ; d leaves Y ; leaves a; ? lordosis . Definition of Behavioural Measures Time together : recorded when the animals were less than 10 cm apart . Time apart : recorded when they were over 10 cm apart . Approach : distance between animals decreases from over 10 cm to less than 10 cm . Leave : distance increases from less than 10 cm to over 10 cm . Approachings and leavings were not recorded if the male and female ran past each other without pausing . Lordosis : this included a graded series of postures from an immobile crouched posture
Observer
A
B I Perspex
®1x2 cm . wire mesh
White pointed hordboord
Fig. 1 . Plan (above) and cross-section along the line A-B (below) . Animals were free to move in the concentric runways and to investigate each other through the wire partition. Clear Perspex walls allowed the observer at position B to see all interactions . Width of runways, 8 . 5 cm ; length of outside wall, 72 cm ; height of walls, 34 cm .
3
with the tail touching the floor to the full lordosis posture with the tail elevated above the general level of the back. Tests During the Oestrous Cycle Twelve animals were tested once on each of days 2, 3 and 4 between 14 .30 and 15 .30 hours. The tests spanned 4 oestrous cycles and were randomized so that the first test could be on any dioestrous day of the cycle . Tests were not necessarily on successive days of the cycle . On day 1 (oestrus) 11 of the 12 animals were tested four times, the tests being separated by 2 .5-h intervals. Testing session began at 09 .40, 12 .12, 14.44 and 17 .15 hours and lasted for 60 min, so that up to four animals could be tested on any one day . Analysis of Data From the measures recorded on the keyboard the following derived measures were obtained for analysis from each 10-min test : Total female approaches 1 . YAp % = Total approaches by male and female Total female leaves 2 . ?L% = 0 Total leaves by male and female . Female Index of Approach = YAp % minus 3 ?L This gives an overall index of female approach or avoidance . It is negative if the female leaves more than she approaches and positive if she approaches more than she leaves (Hinde & Atkinson 1970) . Measures of approaching and leaving were based on the time when both animals were free to interact . If a female showed lordosis in the runway test, the time available for free interaction was less than 10 min . All subsequent analysis is based on time when the female was not in lordosis, i .e . all male approaches and leaves made while the female was in lordosis are dropped from the record . Statistics Non-parametric statistical tests were used to assess the data (Siegel 1956) . Comparisons between the same animals on different days were made using the Wilcoxon matched-pairs test . * No predictions were made about the direction *Comparisons between independent sub-groups used the Mann-Whitney U test .
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
of the differences and two-tailed probabilities were used throughout . Spearman rank correlation coefficients (rs) were calculated to assess the relationship between various measures . The Kendall coefficient of concordance (W) was used to assess the degree of agreement among animals for various measures over the four tests during oestrus . Results The aim of the study was to compare the behaviour of oestrous females with their behaviour on other days of the cycle. For this purpose a single runway test from day 1 was required for each female, which best represented her full oestrous state . The first stage of the analysis seeks to relate measures of lordosis (receptivity) to the runway measures of approaching and leaving . Receptivity During Oestrus Lordosis in arena. See Table I . Arena tests with full access to a male revealed that all 11 females were in oestrus . Five showed lordosis on all four tests, four on three tests, and two on two tests. On test 3 (4 to 5 h after lights off ) all showed lordosis . The median latency to lordosis was shortest on test 3 . The Kendall coefficient of concordance (W) for the four tests was 0 . 30 (P < 0 .01) showing that there was a significant agreement between females on this latency measure . Lordosis in runway. See Table I . Only eight females showed lordosis in the runway, although all showed it on at least two tests in the arena . Of the eight females, five showed lordosis on only one test, one on two tests and two on three tests. Table I . Measures of Lordosis for 11 Oestrous Females Tested at 2 . 5-h Intervals both in the Runway and in the Arena with Full Access to a Male
921
Lordosis duration varied from 11 % of a test to 89 % ; the median longest duration for the eight females was 59 % . One female showed her longest duration on test 1, three on test 2, two on test 3 and two on test 4 . Relation between lordosis in runway and arena . Eight females showed lordosis in the runway ; their median arena latency immediately after they had showed runway lordosis was 3 . 8 s while their shortest arena latency after runway tests without lordosis was 13 . 0 s (Wilcoxon test, P < 0 . 05) . The shortest arena latency for females who showed runway lordosis was 2 . 5 s and for the three females who never showed runway lordosis it was 9 . 0 s (Mann-Whitney U test, P < 0.05) . Lordosis in the two situations correlates well . Animals who show runway lordosis are likely to show a very short latency to lordosis in the arena when tested immediately afterwards . Females who never showed runway lordosis have longer latencies (at best) in the arena than females who show runway lordosis . Relation between lordosis in the arena and SAp %, ?L% and female index. Figure 2 shows the medians of these four measures plotted for the four tests during oestrus . As already shown, females tend to show their lowest latency to lordosis on the same test . Similar Kendall coefficients of concordance (W) are not significant for the runway measures of ?Ap % (0 . 02), 9L% (0 . 14) or female index (0 . 17) ; that is, animals do not show their maximum scores for these measures on the same tests . It follows that
so
FEMALE INDEX - .20
60
0
SECONDS 120
Oestrous test 40
1
2
3
4
No . showing lordosis 6 Median latency (s) 120 No. showing their shortest latency 0
9 28
11 8
10 13
3
4
4
1
5
4
3
1
3
2
2
All tests
Lordosis in arena
20
11/11 0 z OFSTRUS TEST
Lordosis in runway
No. showing lordosis No. showing their maximum lordosis duration
8/11
Fig. 2. Four tests were conducted at 2 . 5-h intervals during oestrus starting at 09 .40 (80 min before lights-off), 12 .12, 14.44 and 17.15 hours. Four measures (medians, N = 11) are plotted for each of these tests . • : latency to lordosis in the arena ; 0, 9Ap% ; o, ?L% ; A, female index .
9 22
ANIMAL BEHAVIOUR, 27,
latency to lordosis in the arena correlates weakly with these three measures . Table II shows that the female index was the most highly correlated, showing a significant negative relationship to lordosis latency on three out of four tests . A comparison of the measures ~Ap %, ?L and female index for tests where females were most receptive and least receptive (measured by latency to lordosis in the arena) show that only female index differs significantly between the two conditions (Table III) . Female Ap differs by only 5 % ; ?L % differs by 16 %, but with a large variance . The net balance between approaching and leaving is thus the best single indicator of potential receptivity . Relation between lordosis in the runway and lAp %, ?L % and female index . For the eight females who showed lordosis in the runway, the median scores for ~Ap %, ?L % and female index were calculated for tests with and without lordosis (Table IV) . The female index was more positive (P < 0 . 05) and ?Ap % was greater (P < 0 . 05) on those tests where lordosis occurred ; ~L % were slightly higher on tests without lordosis but not significantly . Table V shows the most extreme scores for the three females who never showed lordosis and for the eight females who showed some lordosis in the runway. The females who showed lordosis
3
also attained a greater YAp % score (P < 0 .05) and a more positive female index (P < 0 . 05), than did females who never showed lordosis . Females showing lordosis tended to leave less but this difference was not significant . Discussion. The evidence reviewed so far indicates that receptivity increases throughout the day of oestrus and reaches a peak about 5 h after lights-off (cf. Beach et al . 1976) . Measures of approach and avoidance by the female show an overall relationship with measures of receptivity although their variation with the time of day is not nearly so marked . Lordosis in the arena and in the runway are correlated and both relate most consistently to the female index . As the female index becomes more positive, that is as the balance between female approach and female leaving shifts towards relatively more approach, so the females are likely to be more receptive . However, animals vary in the rate at which they attain full oestrous behaviour and tests at each time of day contain animals at different stages of oestrus . As the female index was correlated with latency to lordosis in the arena and was related to the occurrence of lordosis in the runway, the oestrous test with the highest (most positive) female index for each animal was used to compare with the other three days of the oestrous cycle .
Table II . Spearman Rank Correlation Coefficients (rs) between the Latency to Lordosis in the Arena and Three Measures of Female Behaviour in the Runway during the Four Oestrous Tests Oestrous tests Correlation of lordosis latency with ?Ap %
Female index
1
2
- 0 . 29
0 . 18
+0-71 P < 0 . 05 -0-66 P < 0 . 05
-0 . 16 - 0 .23
3
4
- 0 .70 P < 0 .05 -0 .23
-0-42
- 0 . 53 P < 0.1
-0-59 P < 0.1
+0 . 32
Table III. This Shows the Corresponding Median YAp %, ~L % and Female Index for Tests Preceding Each Animal's Highest and Lowest Latency to Lordosis in the Arena Lordosis latency (s)
?Ap %
~L %
Female index
Test with : Lowest latency
6
38
58
- l
Highest latency
120
33
74
- 27
NS
NS
Wilcoxon test
P < 0 .05
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
923
Table IV. Comparison of YAp %, YL% and Female Index between Tests with and without Lordosis for Eight Oestrous Females Median for tests
YAp
YL%
With lordosis
60
57 . 5
+ 19 . 5
Without lordosis
30 .5
65
- 25
P < 0 . 05
NS
P < 0 .05
Wilcoxon test
Female index
Table V. From Four Tests during Day 1, Tests with the Highest ?Ap %, the Lowest 2L and the Most Positive Female Index, for the Females who never Showed Lordosis and those who Showed Lordosis
N Females never showing lordosis Females showing lordosis
Highest ?Ap %
Lowest ?L %
Most positive female index
3
35
66
- 33
8
74 .5
33
+ 23 . 5
NS
P<0 . 05
Mann-Whitney U test
P<0 .05
Tests during dioestrus were conducted 3 . 5 to 4 . 5 h after lights-off, corresponding to the period of maximum receptivity on day 1 . Behavioural Changes over the Oestrous Cycle The next part of the analysis looks at changes over the oestrous cycle . The first section concentrates on changes in the measures of approaching and leaving already described and the relationship between them . In the later sections new measures (derived from the approachleave data) are introduced and described . Relationship between approaches and leaves . Figure 3 shows the group median scores for three measures on each day of the oestrous cycle . The female index, which reflects the balance between approaching and leaving, rises significantly between days 3 and 4 and over dioestrus (days 2 to 4) ; it shows a further marked rise at oestrus . The percentage of female leaves (~L %) falls significantly between days 3 and 4 and over dioestrus ; this trend is continued into oestrus . The percentage of female approaches (SAp %) rises significantly over dioestrus but most between days 2 and 3 ; again there is a marked rise at oestrus . Changes in female index could reflect changes in the proportion of approaches and/or of leaves . It is clear from the graph that both measures change over the 4-day period . The female index correlates strongly with both measures, having an inverse relation-
DAYS 2-4 0 4A % p<0O1 o 9L% p< 0. 001 4 91NDEX P<0.001 9 INDEX *20
I 100
80
0
60
-20
40
-40
20
t
3
4
1
-60 1 0
2
-80
DAY OF OESTROUS CYCLE
Fig . 3 . The medians for YAp %, YL % and Y index are plotted for each day of the oestrous cycle. The same data for day 2 is plotted twice . The significance of differences between adjacent days are shown on the graph : *P < 0 . 05, **P < 0 . 01, ***P < 0 . 001 . The significance of changes over dioestrus (day 2 - day 4) are also shown .
ANIMAL BEHAVIOUR, 27, 3
9 24
ship with YL Y. and a positive relationship with ?Ap % on each of the four days (Table VI) . The different shapes of the two curves representing ?Ap % and ?L % suggest that these two measures are not varying together . This is borne out by the correlations between them which are low and non-significant on each day . Probabilities of approaching and leaving. In a sequence of approaches and leaves by two interacting animals each of the four events male approach, female approach, male leave and female leave, must be preceded or followed by one of two possible events . For example, after a male approach there must follow either a male leave or a female leave . Similarly, before each male approach there must be either a male leave or a female leave . The percentages of male and female moves preceding and following each of the four events named above were calculated for each day of the cycle and are displayed in Fig. 4. These percentages can be interpreted as probabilities of approaching and leaving and will be referred to as probabilities throughout the rest of the paper. Bouts of time together and time apart . The complete 10-min sequence of male-female interaction consists of alternating bouts of time spent together and time spent apart . Each bout of time together (or apart) may be initiated by either sex and ended by either sex . Thus there are eight different possible types of bout in the sequence . In Figs . 5 and 6 are plotted the four types of bout accounting for time spent together and the four types of bout accounting for time spent apart. These show the total time spent in each type of bout as a percentage of total test time .
Each type of bout accounts for a different proportion of the observation time . The time that any one of these eight types of bout contributes to the total test time may be affected by the number of such bouts and/or by their duration . For example, if one type of bout accounts for 30% of the total time, it may be because there are numerous short bouts of this particular type or because there are fewer but longer ones. Figures 5 and 6 show that the two graphs depicting the number of bouts and their total time follow each other closely in most cases . However, both the number and occurrence of these bouts depends on how readily the male and female approach and leave one another. To estimate this parameter mean bout lengths were calculated for each type on each day of the cycle . The shortest bouts were YL-SAp (group mean on day 2 = 4 . 8 s) and the longest were SAp- Sl, (group mean on day 2 17 . 6 s) but there was considerable variation both between animals and between days . In order to assess the changes between days the mean score for each animal was calculated as a percentage deviation from that animal's mean score for all four days of the cycle . Table VII shows whether bouts became longer or shorter over dioestrus, between days 4 and oestrus, or over the whole cycle. It can be seen that the durations of four types of bout change significantly at some stage of the cycle and that changes in one direction over dioestrus are frequently reversed at oestrus . Discussion . The previous analysis has shown that approaching and leaving are not correlated and can vary independently. The percentage of female approaches and the percentage of female
Table VI. Spearman Rank Correlation Coefficients (r,) between YAp %, 9L % and the Female Index for Each Day of the Oestrous Cycle Day of oestrous cycle 2
3
4
1
Y Index : ?Ap
+0-60 P < 0 .05
+0. 62 P<0 . 05
+0 . 64 P<0 . 05
+0 . 84 P<0.001
? Index : YL%
-0-57 P < 0 .05
-0-67 P<0 . 05
-0-77 P<0 . 01
-0-49
+0-28
-0-13
-0-04
NS
NS
NS
rs
correlation between
9Ap% : ?L%
N= 12
NS
0 . 00 NS
N= 11
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
leaves change significantly over the oestrous cycle, but they change at different rates . Changes in the probability of one event (an approach or a leave) occurring with increasing time since the previous event must underlie the other behavioural changes observed in this analysis . The overall probability of a particular bout type ending with time since its initiation, is crudely reflected in its mean length . Changes in bout lengths must affect the number and total duration of bouts of time spent together and apart (Figs . 5 and 6) . Concurrent changes over the cycle in the probability of male approaches (or leaves) and female approaches (or leaves) After a male approach
After a female approach
925
after a specific event will affect the overall probabilities of approaching and leaving shown in Fig. 4 . Mean bout lengths vary widely and it is likely that the length of each bout is affected not only by the immediately preceding leave or approach but by a whole range of events that have occurred previously during the test . Significant changes in probabilities of approaching and leaving (Fig . 4) and in bouts of time spent together and apart (Figs . 5 and 6) fall into three categories . Some changes occur only during dioestrus (days 2 to 4), some occur between day 4 and day 1 (oestrus), while some occur during dioestrus and again at oestrus . After a mal¢ Icovo
After a female leave
1/. dA
°/o
O* A
A N S c/o I
Before a male approach
Before a female approach
+l» Cr L
Before a male leave
+/o
d' A
»/o
A
Before a female leava
250-
2
3
4
1
2
2
3 4 1 2 2 3 4 1 DAY OF OESTROUS CYCLE
2
Fig . 4 . The four graphs on the left show the percentage of female leaves and the percentage of male leaves which either follow or precede all male approaches and all female approaches ; the four graphs on the right show the percentage of female approaches and the percentage of male approaches which either follow or precede all male leaves and all female leaves . Group medians are given for each day of the oestrous cycle . The same data for day 2 is plotted twice. L = leaves ; A = approaches . The following significant differences are shown by asterisks : (1) Asterisks above each graph show significant differences between male and female percentages on any single day, e.g. a male approach on day 2 is significantly more often followed by a female leave than by a male leave (top left-hand graph) . (2) Asterisks on the graph show significant changes in percentage between adjacent days of the cycle. (3) Asterisks on the bar at the bottom left of the graphs show significant changes in percentage between days 2 and 4 (i .e . over the three dioestrous days of the cycle) . *p < 0-05 ; **,P < 0 . 01 ; ***,P < 0 .001 ; Ns = not significant .
926
ANIMAL BEHAVIOUR, 27,
For most measures the extreme points on the graph are on day 1 and day 2 .
not show lordosis in the double runway . Even at the height of oestrus it was possible to collect data on her interaction with the male . Second, the analysis of the approach-leave data revealed very subtle changes in the male-female relationship which could be readily quantified . The lordosis quotient used to measure degrees of receptivity in the female rat is not applicable to hamsters as the female assumes lordosis before the male mounts . Hamster receptivity has been measured as total time spent in lordosis during a test of given length, as latency to the first lordosis, and as duration of the longest-held lordosis (Carter et al . 1973 ; Carter & Porges 1974) . My own observations (unpublished) have shown,that the latency to the adoption of the lordosis posture is dose dependent over a wide range of oestrogen doses (1 to 30 .tg oestradiol benzoate with 500 gg progesterone) . Latency to lordosis is thus likely to be a good measure of intensity of oestrus in the present study. How-
General Discussion The initial aim of this study was to find a way of describing and quantifying male-female interactions; it was hoped to use such a method to show changes in the male-female relationship between dioestrus and oestrus . By concentrating on proximity measures (bouts of time spent together and apart) rather than overt behaviour patterns, it was intended that the same measures could be collected on each day whether or not the females were in oestrus. The type of apparatus used was dictated by a need to prevent the oestrous female from spending most of the observation period in lordosis . This method has been successful in two ways . First, a female in the double runway assumes the lordosis posture less than when she is given free access to a male ; on many oestrous tests females, who were receptive in the arena, did OfA-9L
3
OA -dL
9A-9L
9A- d'L
40CF
CZ':
353025 20 151050-
1
2
3
4
1
i 2
I
2
3
I
1
4 1 2 2 3 4 DAY OF OESTROUS CYCLE
1
Fig . 5 . The two left-hand graphs refer to bouts of time spent together which were initiated by the male, and the two right-hand graphs to bouts of time spent together initiated by the female . Group medians are plotted for each day of the oestrous cycle. The same data for day 2 is plotted twice. --*--, time spent together as a percentage of total test time . --0--, number of bouts of time together as a percentage of total number of all bouts both together and apart . The following significant differences are shown by asterisks : (1) Asterisks on the graph show significant changes in percentage between adjacent days of the cycle . (2) Asterisks on the bars above each graph show significant changes in percentages between days 2 and 4 (i .e . over the three dioestrous days of the cycle) . White bar = % of total time ; black bar = of total number of bouts . Ns = not significant ; *P < 0 . 05 ; **P < 0 . 01 ; ***P < 0 . 001 .
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
ever, it must be remembered that immediately before the test used to measure lordosis latency, the male and female have been together for 10 min in the double runway . This latency measure also has the advantage that the male can be separated from the female before intromission CL-9A
takes place . This eliminates the risk of inducing pseudopregnancy, an important point if daily changes in the subsequent oestrous cycle are to be studied. Lordosis is broadly related to the female index which in turn is based on female approaching
Cf L-CTA
YL-CYA
40c .€7
927
YL-OA
F1.4Y
C€SJ
O
35300
25-
1
201510-
0
5 O
-
~ i
2
c
o
o'
3
1
I
2
2
i
1
1
4 1 2 2 3 4 DAY OF OESTROUS CYCLE
1
2
3
4
Fig . 6 . The two left-hand graphs refer to bouts of time spent apart which were initiated by the male and the two right-hand graphs to bouts of time spent apart initiated by the female . Group medians are plotted for each day of the oestrous cycle . The same data for day 2 is plotted twice . -h, time spent apart as percentage of total test time . --0--, number of bouts of time apart as a percentage of total number of all bouts both together and apart . Significant differences are shown as in Fig . 5 . Table VII . Changes in Bout Lengths between Days of the Oestrous Cycle. Bout Lengths for Each Day Were Expressed as a Percentage Deviation from the Mean Bout Length Over all Four Days . This Table Shows the Difference in this Percentage Deviation Between Days of the Cycle and Indicates whether Mean Bout Lengths Increased by over 10 % (+), Decreased by over 10 % (-) or Changed by Less than 10 % (0) Days 2 to 4 change
Days 4 to 1 % change
Days 1 to 2 % change
Bouts together
dA-dL
-
YA-?L
-
A-dL
-
33 .4 45 . 2 P < 0 . 05 62 . 4 P<0 . 05 63 . 9 P < 0 . 05
+ +
37 . 4 45 .4
0 0
4.0 0.2
+
65 . 4 P<0 .01 41 . 1
0
3 .0
+
22 . 8
9.2
+
9. 1 5.5 49 . 6
0 +
87 . 0 P<0 .01 6.3 38 . 3 16 . 9
Bouts apart
d L-9A
-
SL- o'A YL-9A YL-TA
+
0 96 .2 P < 0 . 001 0 2.8 32 .8 0 66 . 5 -
1
2
928
ANIMAL BEHAVIOUR, 27, 3
and leaving . The female index does not fluctuate in precise synchrony with changes in degree of receptivity, probably because it is affected more by subtle interaction with the male than is lordosis itself. The more quickly a female showed lordosis in the runway situation, the less data on male-female interaction were collected as female lordosis time had to be deducted from the 10-min test data . There is thus a sampling problem which makes it more difficult to collect good approach-leave data from females who are highly receptive . As expected, many changes in the malefemale interaction between oestrus and dioestrus were shown by this analysis ; however, the similarly large changes within the three days of dioestrus were not expected and merit some discussion. A second unexpected finding was that approaching and leaving were not correlated and thus varied independently . This leads to almost infinite variation in the fine details of the male-female interaction as approaching and leaving change at different rates over the cycle . The most obvious independent variable associated with the oestrous cycle is the hormonal state of the female . While this is known to change over the four days, there is no a priori reason to assume that the hormonal state of the male changes also . The observed changes in the male-female relationship can be related to known hormonal changes throughout the oestrous cycle . Baranczuk & Greenwald (1973) measured plasma oestradiol at intervals over the entire cycle . For the first 11 days oestradiol (at less than 20 pg/ml) is barely above the level in ovariectomized females . Towards the end of day 2, plasma levels begin to rise and reach about 90 pg/ml 24 h later . During day 4 there is a further rapid increase (up to 180 pg/ml) which initiates the ovulatory LH surge . After this oestradiol falls rapidly to its lowest level on day 1 ; behavioural oestrus begins after oestradiol starts to decline, and continues (in unmated females) for about the next 16 h while oestradiol is at its lowest . Progesterone levels rise (from about 7 ng/ml) through the second half of day 4 and peak (16 ng/ml) during the dark phase of day 1 while behavioural oestrus is at its height (Norman & Greenwald 1971) . In the present study females avoided the male most on day 2 when oestrogen levels are at their lowest ; my own unpublished observations show that high avoidance is characteristic of ovari-
ectomized untreated females and may thus be a consequence of low oestrogen levels . For a group of eight ovariectomized females tested in the double runway at the same time of day as dioestrous females in the present study, ?Ap was 17 %, 9L % was 79 % and the female index was - 64 .5. These figures are almost identical with those of day 2 females (Fig . 3) . Although female behaviour changes over days 2 and 3 most of the significant changes occur during days 3 to 4 when oestrogen levels are rising rapidly . Further changes occur during days 4 to 1 including the appearance of receptive behaviour. To what extent these behavioural changes can be separately attributed to oestrogen and progesterone is a matter for further study, though in preliminary experiments changes in female approaching and leaving have been obtained with oestrogen alone, as has lordosis, after several days of treatment (unpublished observations ; see also Carter et al . 1973) . Changes in the female's hormonal state could have far-reaching effects on the male-female relationship . First of all, hormone-dependent changes in the female's behaviour could affect the male ; consequent changes in his behaviour could in turn affect the female . Hormonedependent changes in the female which attract her to the male or which attract the male to her could also affect the behaviour of both partners. For example, it is known that olfactory cues from the female attract the male and increase his likelihood of mounting (Murphy 1973 ; Landauer et al. 1978). Both males and females produce ultrasonic calls . Females in oestrus call more than non-oestrous females and the rate of calling of both sexes is increased in the presence of the sexual partner (Floody & Pfaff 1977 ; Floody et al . 1977) . In conclusion, the present analysis is based on measures which were obtained in an artificial situation where the animals did not have free access to one another. Interpretation of the data should be guided always by observations made in a more natural interaction . One advantage of the apparatus used is that the animals can move freely and extensively and the artifacts of testing in a small arena are avoided. By eliminating the opportunity for the display of aggression and most lordosis (except at the height of oestrus) more subtle aspects of the female's behaviour can be revealed and recorded quantitatively . The second advantage is that analysis of a relationship in terms of approaches and leaves allows
STEEL : OESTROUS CYCLICITY IN HAMSTER BEHAVIOUR
as on the other days of the cycle as it does not rely on specific behaviour patterns which may be present on some days of the cycle and not on others . However, it does not preclude other relevant behaviour being recorded and superimposed on the sequence of proximity measures (Steel 1979, in press) . A subsequent paper shows the value of this method in exploring the roles of both partners in the complex and continually changing malefemale relationship during the oestrous cycle (Steel 1979, in press) . REFERENCES Baranczuk, R. & Greenwald, G. S . 1973 . Peripheral levels of oestrogen in the cyclic hamster . Endocrinology, 92, 805-812 . Beach, F . A ., Stern, B ., Carmichael, M. & Ranson, E. 1976. Comparisons of sexual receptivity and proceptivity in female hamsters. Behav . Biol., 18, 473-487 . Bunnell, B . N ., Boland, B . D . & Dewsbury, D. A . 1977 . Copulatory behavior of golden hamsters (Mesocricetus auratus). Behaviour, 61, 180-206 . Carter, C. S . & Porges, S. W. 1974. Ovarian hormones and the duration of sexual receptivity in the female golden hamster. Horm. Behav., 5, 303-315 . Carter, C. S ., Michael, S . J. & Morris, A . H. 1973 . Hormonal induction of female sexual behaviour in male and female hamsters . Horm . Behav ., 4, 129-141 . Floody, O . R . & Pfaff, D . W . 1977 . Communication among hamsters by high frequency acoustic signals. III. Responses evoked by natural and synthetic ultrasounds . J. comp . physiol. Psychol ., 91, 821-830.
929
Floody, O . R., Pfaff, D . W . & Lewis, C. D. 1977 . Communication among hamsters by high-frequency acoustic signals. 11 . Determinants of calling by females and males . J. comp. physiol. Psychol., 91, 807-820. Hinde, R . A . & Atkinson, S . 1970. Assessing the roles of social partners in maintaining mutual proximity, as exemplified by mother-infant relations in rhesus monkeys . Anim . Behav., 18, 169-176 . Johnston, R . E . 1977 . The causation of two scent marking behaviours in female hamsters (Mesocricetus auratus) . Anim . Behav., 25, 317-327 . Landauer, M. R ., Banks, E. M. & Carter, C . S . 1978 . Sexual and olfactory preferences of naive and experienced male hamsters . Anim . Behav ., 26, 611-621 . Murphy, M . R. 1973 . Effects of female hamster vaginal discharge on the behaviour of male hamsters . Behav . Biol., 9, 367-375 . Norman, R . L. & Greenwald, G . S . 1971 . Effect of phenobarbital, hypophysectomy and X-irradiation on preovulatory progesterone levels in the cyclic hamster . Endocrinology, 89, 598-605. Payne, A P . & Swanson, H . H . 1970. Agonistic behaviour between pairs of hamsters of the same and opposite sex in a neutral observation area. Behaviour, 36, 259-269 . Siegel, S . 1956 . Nonparametric Statistics for the Behavioral Sciences. New York : McGraw-Hill . Steel, E . 1979 . Changes in female attractivity and proceptivity throughout the oestrous cycle of the Syrian hamster (Mesocricetus auratus) . Anim . Behav., 27, in press. White, R. E . C . 1971 . WRATS : A computer compatible system for automatically recording and transcribing behavioural data . Behaviour, 40, 135-161 . (Received 23 October 1978 ; revised 6 December 1978 ; MS. number : 1824)