Abstract
Estrous cycle is a physiological change in female individuals that associate with reproduction including rats. It has four stages categorized as proestrus, estrus, metestrus and diestrus. Rats are an ideal animal for investigation of changes occurring during the reproductive cycle due to its short cycle length. The estrous cycle in rats lasts averagely in 4- 5 days and is repetitive. The estrous cycle in laboratory rodents can be useful measure for the reproductive study of estric animals either cytologically or histologically. In cytological evaluation, an analysis of a vaginal smear is often exercised in classifying the epithelial cells to determine the stages of estrous cycle. The fast and simple methodology included the collection from animal’s vaginal lavage followed by slide evaluation and stages determination. The stages of estrous cycle are identified by the absence, presence or proportion of the described basic cell types as well as the density and arrangement of the cells on the slide. Even though different studies involving the estrous cycle using different terms of cells, the result obtained always leading to the similar result which are in proestrus and estrus, the stages are predominated by nucleated cells and cornified cells respectively. Leukocytes most likely to predominate the stage of estrus and diestrus. Additionally, the epithelial cells reflect a developmental continuum which makes them hardly been characterized into several types. The review of the assessment of rodent estrous cyclicity provides details description of its phases, qualitatively and quantitatively. Generally, the observed epithelial cells were consistent with qualitative descriptions of the stages while quantitative analysis revealed subtle differences in the vaginal cell population.
1.0 INTRODUCTION
Reproduction is an important characteristic feature of living organisms because it plays role in regulating population size of organisms (Kendeigh, 1961). Reproduction also acts as a medium for organic evolution by transmitting advantageous variations to the offspring. Semelparity and iteroparity are the two life-history strategies demonstrated by animals and plants. Semelparity is referred to production of offspring once in a lifetime meanwhile iteroparity refers to production of offspring several times during entire life cycle (Angelini & Ghiara, 1984). In aseasonal environment, an iteroparous species reproduce continuously whereas in a seasonal environment, reproduction usually occurs at most favourable period of the year.
Most animals reproduce sexually, nevertheless in a various way. Vertebrates have one of the following three reproductive strategies: ovipary, ovovivipary, or vivipary (Schmidt, 1996). The development of an embryo within an egg but outside the mother’s body is called ovipary. This strategy mostly practiced by reptiles, amphibians and in all aves. Some fish species and reptiles have ovovivipary strategy that allowing embryo to develop in female body until it hatches. Meanwhile, vivipary is the development and sustenance of an embryo within the mother’s body. A period of parental care of the offspring may be exercised after the animal are giving birth. This reproductive strategy practiced by almost all mammals (Schmidt, 1996).
In mammals, there are two types of reproductive cycle which are menstrual cycle and estrous cycle. Menstrual cycle is a reproductive cycle known for primates, apes and human while estrous cycle is a main reproductive cycle of females of other non-primate mammals. For instances, rats, mice and horses. There are also a variety of different estrous forms which are monoestrous, polyestrous and seasonality polyestrous. Monoestrous animals have one estrous cycle per year such as dog, foxes and bear. Seasonality polyestrous categorized the animals which have multiple estrous cycle only during certain periods of the year such as sheep and horses. Animals that cycle throughout the year is categorized into the polyestrous animals such as rats, mouse, cattle and pigs.
Rat (genus Rattus) generally grouped the members of several rodent families that having bodies longer than about 12 cm. Rodents are mammals that are part of the Rodentia order under the Mammalia class. They are known for reproducing rapidly because they cycle throughout the year and breed year- round. Female rodents are polyestric which show spontaneous ovulation and have regular and consecutive estrous cycle (Paccola et al., 2013). Annually, 5- 10 litters are produced by a single female of rat. At approximately 30 days of age, each litter that consists of 5-6 young reach their sexual maturity and are able to reproduce. As they produce a great number of young, they also have high mortality rate. That is because most small mammals weighed under 1 kg are characterized by rapid reproduction and high mortality which means for animals of the same size with higher birth rates must have correspondingly higher death rates (Sibly & Brown, 2009).
Estrous cycle is a physiological change in female individuals that associate with reproduction including rats. It has four stages categorized as proestrus, estrus, metestrus and diestrus (Marcondes et al, 2002). Rats are an ideal animal for investigation of changes occurring during the reproductive cycle due to its short cycle length (Marcondes et al., 2001). The study aims to identify the stages of estrous cycle in small mammals especially rats based on different types of epithelium cell in vaginal smear samples. Analyzing a vaginal smear is largely an exercise in classifying the epithelial cells to eventually determine the stage of estrous cycle. Rats often use in scientific usage and information obtained from this study probably can be used to understand breeding ecology and reproductive pattern of other animals too. Furthermore, evaluations of vaginal cytology in the laboratory rodent can be more useful in humans to determine the functional status of the reproductive system especially in menses, alterations in vaginal pH, cervical mucous viscosity and blood hormone levels (Goldman et al., 2007). The assessment of rodent estrous cyclicity can offer insight into potential adverse effects in humans.
2.0 LITERATURE REVIEW
2.1 Estrous Cycle
In Heape’s (1900) publication, the term estrus was first used. Estrus defined as the special period of sexual desire in the female. A Greek word oistros which means madness or frenzy has been adapted into Estrus which refers to the period of heat or sexual receptivity (Goldman et al., 2007). By making the Estrus as a base word, Heape (1900) defined the rest of the stages as proestrus, metestrus and diestrus.
In Goldman et al. (2007) and Westwood (2008), the authors documented that morphological changes in ovaries, uterus and vagina, characterized the stages of estrous cycle. In proestrus stage, an animal is coming into heat while in metestrus is a short period where conception is absent and diestrus is a period of short rest where reproductive tracts prepare for receipt of the ovum (Heape, 1900). The ovulation occurs from the start of proestrus stage to the end of estrus stage (Young et al., 1941; Schwarts. 1964). The mean cycle length in the female rat is 4 days, predicted from the onset of sexual maturity up to the age of 12 months (Long & Evans, 1922; Freeman, 1988).
The proestrus and estrus phases last for 12 hours respectively while metestrus lasts for 21 hours and diestrus lasts for 57 hours (Paccola et al., 2013). However, Long and Evans (1922) classified the estrous cycle in five phases. The phases are termed as proestrus, estrus, metestrus I, metestrus II and diestrus (Gronroos & Kaupilla, 1959). Lasted about 15- 18 hours, the stage of metestrus I was also known as early metestrus while metestrus II or known as late metestrus lasts for 6 hours (Hebel & Stromberg, 1986). Maeda et al. (2000) and Westwood (2008) recorded the cycle has also been divided into 5 phases as proestrus, estrus, diestrus I or metestrus, and diestrus II. In a 4- day cycle and 5- day cycle, diestrus lasts for 2 days (diestrus 1 and diestrus II) and 3 days (diestrus I, diestrus II and diestrus III) respectively (Maeda, 2000). Review by other authors and publications, they considered the additional phase as anestrus stage which demonstrated the inactive ovarian activity (Westwood, 2008).
The ovulation occurs from the start of proestrus stage to the end of estrus stage (Young et al., 1941; Schwarts. 1964). The mean cycle length in the female rat is 4 days, predicted from the onset of sexual maturity up to the age of 12 months (Long & Evans, 1922; Freeman, 1988).
2.2 Vaginal Cytology
The type of cells present in vaginal smears has long been used to identify the stages of the estrous cycle in laboratory rats and mice by microscopic evaluation in aiming to indicate the functional status of the hypothalamic- pituitary- ovarian axis (Cora et al., 2015).
Before the establishment of vaginal cytology, the stages of estrous cycle were defined and characterized based on behaviour changes, for instance when animal accept or ready to copulate. Apart from that, record in Cora et al. (2015) said that the morphological and histological changes can be observed in the reproductive tract of cycling females. Additionally, the observation is made in macroscopic changes such as swelling in the vulva, congestion in the uterus and vaginal excretions such as haemorrhage and mucous. The microscopic changes in uterine tissue and ovaries is observed as well to characterize the stages of estrous cycle (Cora et al., 2015).
However, in small rodents like rats, the macroscopic observations were not always reliable. Tissue pathology was and still is an invasive procedure for staging in-life estrous cycle. The need of more reliable and non- invasive protocol to determine the stages of estrous cycle has led to Stockard and Papanicolaou (1917). This publication was setting up the characterization of vaginal canal changes during the estrous cycle that can be used for any animal species, macroscopically (histology) and microscopically (cytology).
Also from the findings, other researchers such as Joseph Long, Herbert Evans and Edgar Allen, pursued their works and researches and described the estrous cycle of rats of mouse (Long & Evans, 1922; Allen, 1922). Long & Evans (1922) in their publication, expressed the importance of Stockard and Papanicolaou (1917) publication articulately. Developed by Papanicolaou, the Pap smear test now used as a screening test for cervical cancer. In Vilos (1998), the Pap smear is recognized as the most significant advancement in controlling the cancer in the 20th century.
2.3 Cell Type of Vaginal Smears
A sample of cells obtained from the surface of the vaginal epithelium can be used to determine the stage of estrous cycle by viewing at low microscopic magnification (Long & Evans, 1922). They both also recorded that the layers of vaginal mucosa include the stratum corneum, stratum granulosum, rete mucosum and stratum germinativum. As the cells shed off, the vaginal epithelium changed morphologically responded to the cyclic differences in vaginal cytology.
The stages of estrous cycle are identified by the absence, presence or proportion of the described basic cell types as well as the density and arrangement of the cells on the slide. Most cells seen in a normal vaginal smear are vaginal epithelial cells. In some protocols, the vaginal smears have been dry fixed and stained with metachromatic stain to clearly characterize and recognize the type of cells present in the smears.
Many publications and authors came out with different type of cells but indicating to the description of the cells, they were looking at the same cell type but with different names. In Hubscher et al. (2005) and Marcondes et al. (2002), the authors described the epithelial cells as leukocytes, nucleated cells and cornified cells. A round cell with nucleus is named as nucleated cells, an irregular shaped of cell without nucleus is named as cornified cells and leukocytes is a little round cell (Marcondes et al., 2002). Leukocytes are very small, round shape and possess multilobulated nuclei. These cells are also known as neutrophils or polymorphonuclear cells.
On the other hand, Paccola et al. (2013), described 5 types of cells in their works as small basophilic cells (SBC), large basophilic cells (LBC), nucleated acidophilic cells (NAC), enucleated acidophilic cells (EAC) and pre- acidophilic cells (PAC). Basophilic cells were represented less distinct cells from basal layer and intermediate epithelial layers. SBC are rounded shape cells with large nuclei and sparse chromatin while LBC are a polygonal shape cells, displayed condensed chromatin and smaller than SBC. Acidophilic cells are from superficial layers and more distinct. NAC are polygonal in shape and had small nuclei while EAC are keratinized cells without nucleus as they moved to the superficial layers. PAC are enucleated small cells and polygonal in shape that are partly keratinized.
On top of that, these three remarkable publications had improvised the protocols of staging and identifying of cells in vaginal smears. NAC, EAC and PAC descripted as cornified cells while on the other hand SBC and LBC descripted as nucleated cells. Paccola et al., (2013), the most recent publication had revised and improvised the type of cells specifically to characterize the stages of estrous cycle and provide insight of identification protocols.
The estrous cycle is divided into 4 stages which are termed as proestrus, estrus, metestrus, and diestrus. The cycle regularly repeats every 4 to 5 days unless interrupted by pregnancy, pseudopregnancy, or anestrus. The cycle has been described in detail elsewhere in Hubscher et al. (2005), Cora et al. (2015) and Goldman et al. (2007).
According to Cora et al. (2015), the evaluation of subsequent vaginal cytology smears has been long used as a tool for the assessment of the estrous cycle in laboratory rats. Collection and preparation of vaginal cytology samples is an essentially non- invasive procedure. Furthermore, the protocols are easily mastered but only with a proper training and continuous exercise as well as practice. The evaluation of the cytology smears must be utilized from well- established- standardized criteria to provide a reliable and consistent data generated from vaginal cytology studies. Smear review and the data obtained can only be as “good” as the technical expertise and experience of the elevator performing the microscopic examination.
Over the past few decades, there are so many works involving the progression of estrous cycle in rats. Year by year, the researchers keep improvising the protocols either histologically or cytologically. From quantitative protocol to qualitative protocol, those are made to improvise the accuracy of getting the information that might be useful for further study. Nevertheless, the results obtained at the end are almost meet the result from one study to another study, proving that they exercised and revised the protocols used. From external morphology to internal morphology, the sparks induced by Stockard and Papanicolaou in 1917 has been very useful to the ones working in this field.
A quantitative and qualitative analysis of the rat estrous cycle has been showed in the present study, including a description of the transitional periods. A detailed description about the presence of epithelial cell types in the smears is also recorded. Additionally, most of the authors performed a statistical analysis to confirm the relevance of the differences of the estrous cycle phases. A detailed classification of the estrous cycle is essentially used in a study about preimplantation and just implanted embryo. Apart from that, these studies provide an insight of the different strategies use in the estrous cycle analyzation that has been described in the literature in many studies and publications. These studies from past decades until present could be possibly contributed to researchers whose studies rely on detailed information about the rat estrous cycle (Paccola et al., 2013).
Originally published 15.10.2019