The Phases of the Oestrous Cycle in the Adult White Rat

Observations were made on thirty-nine adult virgin females derived from ten litters belonging to an inbred colony of albino rats. They were 98-186 days old at the start of the experiment. Each litter-group was housed in a separate cage, and supplied with food and water ad lib.

Daily vaginal smears were taken for 83–88 days, using a loop of nickel-chromium wire heated in a flame and cooled in saline. The smears were fixed in a modified Schaudinn’s solution, consisting of equal volumes of absolute alcohol and saturated mercuric chloride solution. After rinsing in 90% alcohol containing a few drops of iodine in potassium iodide, and then in water, the preparations were stained in Mann’s eosin-blue solution.

To allow of more detailed histological examination, the following technique was developed. After the usual fixation, the smears were stained for 5 min. in acidified 1 % Ponceau-de-xylidine red solution, rinsed in distilled water acidified with acetic acid, and then differentiated in 1% phosphomolybdic acid. They were then counter-stained in 2–5% methylene blue for 1 min. and rinsed in tap water. The smears were either examined wet, or dehydrated, cleared and mounted in Canada balsam. This technique stains acidophil cytoplasm red, and basophil cytoplasm blue. Although the differentiation is not as clear as with trichrome stains (Hartman, 1944; Shorr, 1941), the method is much more rapid and can be used for routine purposes.

All the stained smears were stored and subsequently re-examined in order to check the diagnosis of the following six stages of the cycle, based on published classifications (e.g. Astwood, 1939):

Early dioestrus: thick smear consisting almost entirely of leucocytes; some cornified cells, Shorr cells and a few basophilic epithelial cells.

Dioestrus : thin smear, consisting of leucocytes, basophilic epithelial cells undergoing vacuolation, practically no cornified cells, diminishing numbers of Shorr cells, and Hartman’s type V ‘phagocytozed’ cells.

Late dioestrus: leucocytes, some vacuolated and some clearly nucleated basophilic epithelial cells, no cornified cells, no Shorr cells.

Early oestrus: thick smear consisting of large numbers of hexagonal basophilic nucleated epithelial cells, often disposed in a honeycomb pattern. Towards the end of this phase, the cells separate and cornification sets in.

Oestrus: all, or almost all, the epithelial cells have lost their nuclei and have become cornified. Towards the end of this phase, the smear becomes ‘cheesy’.

Late oestrus : the smear is similar to the oestrous smear, but, in addition to many cornified cells, there are some large basophilic Shorr cells, and some small basophilic epithelial cells. As soon as the leucocytes appear, late oestrus merges into early dioestrus.

Smears were often diagnosed as being intermediate between two of the above stages, and thus twelve were recorded (e.g. L.D./E.O., L.O./E.D., etc.).

For a check on diagnosis, 1100 smears were selected at random and re-examined, and the two sets of data compared.

About a fortnight before autopsy, the mean length of the cycle was calculated for each animal, and the date at which it would be at a preselected stage of the cycle estimated. Starting 3 days before autopsy, smears were taken twice daily, as nearly as possible at 12-hourly intervals, so that the precise stage at which the animals were killed was known. As the greatest changes in the accessory reproductive organs are known to occur between late dioestrus and oestrus (Astwood, 1939), twenty-eight out of the thirty-nine animals were killed within this period. The times of autopsy of the remaining eleven animals were distributed over the other stages of the cycle.

When all the smear records were complete, the mean interval between successive phases of oestrus (i.e. the cycle length) was calculated for each animal. The relative lengths of the various stages were determined by Astwood’s (1939) method. For each animal, the number of days on which each of the six stages of the cycle was recorded was expressed as a percentage of the total period of observation (i.e. 83–88 days). The mean cycle length (in hours) for each individual rat was then subdivided into its six phases according to the ratio of these percentages. Means for litter-groups and a weighted mean for the whole series were then calculated.

The animals were weighed and killed with chloroform within 5 min. of the final smear. The ovaries, adrenals and uteri were dissected out and weighed before fixation. The uteri were weighed twice—first with the fluid in the lumen, and then after the fluid had been drained.

Standard statistical procedures (Fisher, 1946) were used. Comparisons were made by t-tests, by the χ² test and by analyses of variance.

Regularly recurring oestrous cycles were observed in thirty-one of the thirty-nine animals. In the remaining eight animals, oestrous cycles occurred for only part of the time, and were interrupted by periods of dioestrus which varied between 11 and 60 days. Leucocytes were rarely absent from the vaginal smears of these animals. Subsequent observations showed that the irregularity of the oestrous cycles of these rats was due to some pathological condition of the reproductive organs. Those cycles in the eight irregular animals in which the period of dioestrus did not exceed 7 days were regarded as being within the normal range and were included in the analysis.

Daily smears relating to a total of 659 ‘normal’ cycles (over 3000 smears) were examined. The preparations showed all the cell types already described by Hartman (1944) and Jaworski (1950).

The mean duration for the 659 cycles was 4·4±0·04 days. Analysis showed that the variance in cycle length is significantly less within than between individual rats (P<0·001), both in the present series of data and in that published by Long & Evans (1922). Litter mates tended to cycle more consistently than unrelated rats (P≏0·05). There was no significant difference in cycle length within as compared to between litter-mates in eight out of the ten litters (Table 1).

The mean cycle length of the present series (4·4 ± 0·04 days) is significantly shorter than that computed from Long & Evans’ data (5·6±0·08 days; P<0·001). The mode, however, is 4 days in both sets of observations. The two colonies differ in so far as the frequency cycles of 4 days and less is significantly higher in our series (P<0·001); the frequency is significantly lower above 4 days (P<0·001). In both series the distribution around the mode is skew, the incidence of cycle lengths of more than 4 days being greater than for cycles of less than 4 days (Table 2).

Table 3 shows that the variance in the duration of dioestrus and late dioestrus is significantly less between fittermates than between unrelated rats (P<0·001). With the exception of late oestrus, which is on the border-line of statistical significance (P=0·01–0·01), the remaining stages vary as much within, as between, litter-groups.

This suggests that differences in the length of the whole cycle in different animals are due to variation in the duration of dioestrus, and that the duration of the oestrous phase is more or less constant.

Fig. 1 shows the relative duration of the phases of the cycle as reported by Long & Evans (1922), Astwood (1939), Jaworski (1950), and as determined in the present investigation. The figures of Long & Evans and Jaworski are based on the study of smears taken every few hours, while those for the other two studies are indirectly based on the frequency with which different stages were observed in daily smears. It is clear that the relative duration of the different phases is very similar in the four series of data. Long & Evans observed that while the mean duration of oestrus is 38 hr., the mode is 27 ; the higher value of the mean is due to the fact that a number of their rats remained in oestrus for prolonged periods. In view of Astwood’s (1939) later finding, it seems probable that the prolonged cornification noted by Long & Evans in some animals was due to too frequent smearing. It is also possible that the colony included some rats in which persistent oestrus occurred spontaneously (Everett, 1939).

The difference between the present observations and those of Long & Evans in the duration of dioestrus, early oestrus and metoestrus were not statistically significant (P= 0·5–0·3, 0· 5–0·3 and 0·7–0·5 respectively).

Of 1100 smears selected at random and re-examined without reference to the previous recorded diagnosis, 653 diagnoses were in complete agreement; 244 differed by stage; 156 differed by 1 stage and 47 by stages. There were no disagreements of 2 or more stages. Differences in diagnosis were inconsistent in direction so far as the stages of the cycle were concerned. The mean error was 0·3 stage per reading. It would probably have been smaller if reference had been made, for purposes of guidance, to the diagnosis of the preceding days’ smears.

So far as we are aware, there is no previous published record of a systematic check on the accuracy of diagnosis of vaginal smears.

The animals were fully grown, and at autopsy ranged from 183 to 273 days in age. Body weights varied from 156 to 242 g. (mean 200 g.) and appeared not to bear any constant relation to age.

An analysis of variance showed that the variance in body weight was significantly less within than between litter-groups (P≏0·001). Variance in body weight within and between stages of the cycle was not significantly different (P = 0·2–0·1). This suggests that body weight does not fluctuate consistently in phase with the oestrous cycle.

The mean ovarian weight for the thirty nine rats was 61·2 mg. (range 37–88·5 mg.). Five of the eight rats which had been in persistent dioestrus had small ovaries (38·5–47·0 mg.).

The variance in ovarian weight did not differ significantly within and between litter groups (P≏0·1), but the variance within litter-groups was the smaller.

The variance in ovarian weight in animals at the same stage of the cycle tended to be less than that in animals at different stages of the cycle (P= 0·1–0·05). Rats killed between late dioestrus and oestrus had heavier ovaries (mean 62-9 mg.) than those killed at the remaining stages (mean 56·9 mg.). A plot of the frequency distributions showed that in animals killed between late dioestrus and oestrus, both the mean and the mode had shifted to the right, as compared with animals killed at other phases of the cycle. The difference in mean failed, however, to reach statistical significance.

The variance between the right and left ovaries of the same individual did not differ significantly from that between right and left ovaries of rats at the same stage of the cycle (P=0·2–0·1), but it was significantly less that that between litter-mates and between rats at different stages of the cycle (P <0 ·001).

The mean adrenal weight for the series was 65·1 mg. (range 40–94 mg.). Three of the eight animals that had been in persistent dioestrus had low adrenal weights (40, 50 and 50·5 mg.).

It has been shown by Andersen & Kennedy (1932) and Bourne & Zuckerman (1940) that the adrenal glands are heavier during oestrus than at other times. A comparison of the means of the weights recorded in the present study showed again that the glands tended to be heavier between late dioestrus/early oestrus and oestrus than at other times; the differences, however, were not significant statistically (P= 0·1–0·5). A plot of the frequency distributions of adrenal weights during the oestrous phase compared to those in other phases showed that both the mean and the mode of the adrenal weights have shifted to the right at oestrus.

An analysis of variance, on the other hand, showed that the variance in adrenal weight did not differ significantly within and between stages of the cycle (P = 0·1–0·05). This result, like the corresponding one on the ovary, is not, however, inconsistent with the thesis that the weights of the ovaries and adrenals vary in phase with the oestrous cycle.

Further analyses of variance showed that the variance in adrenal weight did not differ significantly within and between litter-groups (P= 0·1–0·05); the variance between right and left adrenals of the same individuals was significantly less than that within and between groups of animals at the same stage of the cycle, and within and between litter-groups (P<0·001).

The weight of the uterus ranged from 206 to 1370 mg. before any fluid was drained from the lumen, and from 206 to 760 mg. after draining. The two lowest uterine weights (206 and 223 mg.) were recorded in animals that had been in persistent dioestrus.

The manner in which the uterine weight varies in phase with the oestrous cycle is shown in Fig. 2. When the uteri were weighed with the fluid in the lumen, the variance within stages of the cycle was significantly lower than that between stages (P = 0·01–0·001); the difference was not significant after the fluid had been drained (P = 0·2–0·1). Fig. 2 shows however, that the weight of the uterus without fluid is higher at oestrus than at other times. This observation agrees with previous reports (e.g. Astwood, 1939).

In both analyses, variance in uterine weight did not differ significantly within and between litter-groups (P>o·2).

The analysis of the data collected in the present study, as well as that of Long & Evans (1922), indicates that variation in the length of the oestrous cycle in the adult white rat is greatly influenced by the genetic make-up of the individual. It also shows that the duration of oestrus is much more constant than that of dioestrus, which consequently has a greater influence on the variability of the whole cycle. These findings point to the advantage of determining the mean cycle length of individual rats before they are submitted to experimental procedures designed to elucidate some aspect of reproductive physiology.

Apart from these new observations, and the check made on the reliability of the diagnosis of vaginal smears, the present study confirms the results previously published by Long & Evans (1922), Astwood (1939), Hartman (1944) and Jaworski

The duration of the oestrous cycle in thirty-nine adult rats derived from ten litters was 4·4 ± 0·04 days.

The variance in cycle length tended to be less within than between litter-groups (P≏0·05). It was significantly less for individual rats than between individuals (P <0·001).

The mean duration of early oestrus was 18 hr., oestrus 25 hr., late oestrus 5 hr., early dioestrus 24 hr., dioestrus 28 hr. and late dioestrus 7 hr. The length of the dioestrous phase is variable and determines the length of the cycle, while the duration of oestrus appears to be relatively constant.

The figures have been compared with those published by other laboratories.

Analyses of variance did not disclose any significant fluctuation in the weight of the body, adrenals or ovaries in phase with the oestrous cycle. The uterus showed a characteristic increase in weight immediately before the onset of oestrus.

The author wishes to thank Prof. S. Zuckerman for his valuable help and advice.