The importance of laboratory animals cannot be overlooked in the field of biomedical sciences. The lab animals are used as animal model in various trials to observe the effect of medicine/hormone/vaccine/toxin trials or for antibodies production
etc. Small rodents like mice and rats are more preferred for animal experimentations than larger animals like dogs, cats, pigs and monkeys (
Amin et al., 1996). Laboratory animal experimentation in animal reproduction requires accurate knowledge of duration and phases of estrous cycle. Only a few numbers of studies were conducted on estrous cycle of rats. The scanty literature available on this topic suggests that the phases of estrous cycle of rat are same as in cattle/buffalo with variability in duration of reproductive phases. The rats have been reported to have proestrus (14 hours), estrus (24-48 hours), metestrus (6-8 hours) and diestrus (48-72 hours) phases in their estrus cycle with cycle duration of 4-6 days (
Auta and Hasan, 2016). There are several methods to assess the estrous cycle in rats which includes vaginal cytology (
Ojeda and Andrews, 1981;
Mclean et al., 2012;
Barrett et al., 2016), biochemistry of urine (
Singletary et al., 2005), visual assessment of vulva/vagina (
Auta and Hasan, 2016,
Barrett et al., 2016) and vaginal wall impedance (
Rodriguez et al., 1997;
Singh et al., 2013). There is high variability in efficacy of these methods in phasing of estrous cycle in rats. Therefore, the present study was conducted with the aim to standardize the phases/duration of phases of estrous cycle in rats by vaginal cytology and visual assessment.
The experiment was conducted in 2022 at the Department of Veterinary Gynecology, College Of Veterinary And Animal Sciences, Sardar Vallabhbhai Patel University of Agriculture and Technology, Modipuram, Meerut, U.P., India. The guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA’s) were followed for experimentation on laboratory animals. The experimental study was duly approved by Institutional Animal Ethics Committee (IAEC) vide no
. IAEC (BMRL/AD/CPCSEA/IAEC/2022/1).
A total of 21 Wistar albino (which had completed their pubertal stage >4 weeks) female rats weighing approximately 100- 200 g were maintained for experimental period at 24±3°C temperature and 55±5% relative humidity with 12-hour light/dark cycle. They were given standard pellet feed and ad libitum water during the experimental period.
Visual assessment is a noninvasive, simple, fast, less stressful technique that can be performed anywhere for phasing of estrous cycle. During visual assessment, the rat was held in left hand and laid in the restraint as forepaws resting on surface, the tail was gently lifted and the vulva was examined. The appearance of vagina at various phases of estrous cycle was recorded and summarized in Table 1.
For vaginal cytology, the rat was restrained in one hand by holding it from the neck dorsally and uplifted to ventral side upward. The tail was grasped by finger of the same hand. The cytology was performed as per the method described by
Cora et al., (2015). Briefly, the vaginal cells were flushed by gently introducing 100 μl of distilled water using micropipette. The distilled water was released over vagina and drawn back into the tip. It should be repeated 4 to 5 times in the same micropipette. Care was taken to avoid penetration of pipette tip in the vaginal orifice. The vaginal fluid containing a few drops of cell suspension was placed on glass slide and air dried. Crystal violet stain 0.1% was used to stain the air-dried methanol fixed vaginal smear. The slide was examined under a light microscope at 10x magnification. Vaginal cytology was performed in 21 rats thrice daily along with visual assessment.
Visual assessment for phasing of estrous cycle in wistar albino rats
In random assessment by vaginal cytology, 8 out of 21 (38.09%) rats were found in proestrus but on visual assessment, vaginal opening was present only in 2 out of 21 rats (9.52%) and partial vaginal opening was noticed in 6 out of 21 (28.57%) rats (Table 2, Fig 1a). Previous workers also reported that during proestrus, vaginal gaping was seen and tissues were moist, reddish pink in color along with striations on dorsal and ventral lips of vagina (
Champlin et al., 1973). So, the findings of partial vaginal opening during proestrus might be due to slight early sampling. A total of 4 of 21 rats (19.05%) were observed in estrus based on vaginal cytology but on visual assessment, vaginal opening was present only in 2 (9.52 %) rats (Table 2, Fig 1b). The partial vaginal opening in 2 rats might be due to slight delay in sampling. Six rats (28.57%) were observed to be in metestrus phase by vaginal cytology, but vaginal opening was present only in 3 (14.29%) rats (Table 2, Fig 1c). Three rats were found in diestrus by vaginal cytology and slight vaginal opening was present in 2 rats on visual assessment (Table 2, Fig 1d). The findings agreed with visual assessment reports of
Champlin et al., (1973);
Auta and Hassan, (2016);
Barrett et al., (2016) and finding of vaginal cytology of
Cora et al., (2015) reported earlier.
Vaginal cytology for phasing of estrous cycle in wistar albino rats
The different stages of estrous cycle were identified by the presence, absence, or proportion of vaginal epithelial cell types and their density. Proestrus stage was confirmed by the presence of small, round, nucleated epithelial cells. Epithelial cells were seen individually throughout the smear. Generally, no neutrophils were observed but occasionally neutrophils were seen in some smears examined. Some smears revealed the presence of both types of cells,
i.e., nucleated epithelial cells in majority and a few non nucleated cornified epithelial cells were also observed (Fig 2a). Similar findings were reported during proestrus stage by
Cora et al., (2015). In our trial, the average duration of proestrus was recorded to be 24 hours which is contradictory to the finding of previous workers (
Allen, 1922;
Long and Evans, 1922;
Astwood, 1939 and
Mandl, 1951) who reported that the average duration of proestrus was 14 hours. The estrus phase was characterized by the predominance of anucleated keratinized epithelial cells, while occasionally large nucleated epithelial cells were also observed in some smears (Fig 2b). These findings were in accordance with
Cora et al., (2015), as they reported abundant anucleated cornified epithelial cells during the estrus phase. Abundant anucleated cornified epithelial cells during estrus probably due to highly increase concentration of estrogen are responsible for “cornification” of epithelial cell. The duration of estrus phase of estrous cycle was recorded between 24 to 36 hours. The duration of estrus in our study shows similarity with the report of previous researcher (
Allen, 1922;
Long and Evans, 1922;
Astwood, 1939;
Mandl, 1951 and
Champlin et al., 1973), who reported average estrus duration of 24-48 hours. Metestrus stage was ascertained by the appearance of large number of neutrophils throughout the smear along with small number of anucleated keratinized epithelial cells. In some smears both nucleated and anucleated epithelial cells and large numbers of neutrophils were also recorded (Fig 2c). The metestrus duration was recorded to be less than 12 hours which agreed with the findings of previous workers (
Allen, 1922;
Long and Evans, 1922;
Astwood, 1939 and
Mandl, 1951) who reported average metestrus duration of 6-8 hours. In vaginal cytology smear, neutrophils were seen in highest number along with moderate number of anucleated keratinized epithelial cells. However, a few small and large nucleated epithelial cells were also seen. These findings were in accordance with the findings of
Cora et al., (2015), who reported that large number of neutrophils and small number of monogranular and anucleated cells were seen during metestrus. Diestrus stage was confirmed by the presence of neutrophils predominantly with few nucleated epithelial cells (Fig 2 d). The duration of diestrus was recorded to be 36-48 hours in the present study which agrees with the findings of
Allen, (1922);
Long and Evans, 1922;
Astwood, 1939 and
Mandl, (1951). Neutrophils were predominant cells in vaginal cytology during diestrus. However, nucleated epithelial cells were seen in moderate number along with few cornified epithelial cells. The finding of vaginal cytology during diestrus in the present study was like
Cora et al., (2015) and
Auta and Hasan, (2016). The duration of proestrus, estrus, metestrus and diestrus were recorded to be 24, 24-36, 12 and 36-48 hours, respectively.
