J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 68-72. https://doi.org/10.47277/JIRB/8(4)/68  
Determination of the Ovulation Time in the  
Laboratory Rats (Rattus norvegicus)  
1
2
2
3
Joy Iyojo Itodo *, Agnes Ifeoma Nwannenna , John Shiradiyi Bugau , Kenneth Owoicho Abah ,  
4
4
3
Danjuma Friday Audu , Grace Imaben Opaluwa-Kuzayed , Simon Azubuike Ubah , Mohammed  
5
2
Babashani , Kuje, Althea Agbi  
1
Department of Animal Science Faculty of Agriculture Federal University of Lafia, Nasarawa, Nigeria  
2
Department of Theriogenology and Production, Faculty of Veterinary Medicine, Ahmadu Bello University Zaria, Kaduna Nigeria  
3
Department of Theriogenology, Faculty of Veterinary Medicine, University of Abuja, F.C.T. Nigeria  
4
Department of Theriogenology and Production, Faculty of Veterinary Medicine, University of Jos, Plateau, Nigeria  
5
Veterinary Teaching Hospital, Ahmadu Bello University Zaria, Kaduna Nigeria  
Received: 10/08/2020  
Accepted: 29/10/2020  
Published: 20/09/2020  
Abstract  
The rat has been elected as the main animal model in different studies involving reproduction. However, there are scarce and  
conflicting data related to its estrous cycle. The aim of the experiment was to determine the time of ovulation in the primiparous  
laboratory rats (Rattus Norvegicus) by counting the number of graafian follicles present in the ovary at the time of oestrus, determining  
the percentage of these follicles that eventually ovulate and determining the 'spread' of ovulation during the oestrus period. Fifty (50)  
albino laboratory rats were observed in oestrus with the help of males to determine the time the female first stood to be mounted. This  
time was considered the onset of oestrus and used as a landmark for timing of oestrus period. From this onset of oestrus, the period was  
divided into 10 x 1-hour intervals into which the female rats were grouped. Each hour interval had five rat members. Ovaries of rats  
harvested at the end of their group-hour intervals were studied histologically, for functional structures. Observed structures were counted  
for the calculation of ovulation rates. All oestrus rats had far shorter periods, the ovulation in this study were found to be widespread  
from the onset to the tenth hour peaking maximally at the sixth and seventh hour of oestrous. Follicular activity was found to be more  
in the left ovaries while ovulatory activity was more in the right ovaries. It was concluded that rats presented far shorter oestrus periods  
and all ovulations in the albino rats used in our laboratory occurred at the onset of oestrus and that the mechanism responsible for the  
first "stance for mounting" is responsible for the ovulation and that ovulation is induced in these rats.  
Keywords: Corpora lutea, Histology, Rats, Estrous, Ovulation  
Introduction  
The rat has been elected as the main animal model in  
several studies involving reproduction. However, there are  
scarce and conflicting data related to its estrous cycle. It  
comprises phases characterized by different cell types in  
vaginal smears (proestrus, estrus, metestrus and diestrus) (1).  
Ovulation is the release of matured eggs from the ovaries. It is  
the culminating effect of the event of the oestrous cycle which  
is the rupture of the follicle and the shedding of the ovum (2).  
Since the laboratory rat (Rattus norvigecus) is the most used  
animal in various scientific researches worldwide especially in  
reproductive analysis, the improvement of its reproductive  
results and fertility rates seems to be a worthy objective. The  
basic principles for detection of ovulation in warm climates are  
similar to those for cool or temperate climates (3).  
description of the behavioural characteristics known as  
oestrous ('heat') should be provided in the studies of ovulation.  
Studies have shown that in rats as well as other animals,  
conception will only occur in those animals that are ovulating.  
Detection of the onset of ovulation, the seasonal patterns of rats'  
reproduction and breeding behavior are remarkably varied,  
therefore, it is very important to achieving high conception  
rates. However, detection of the onset of ovulation is not very  
easy, particularly in the tropics (4).  
Therefore, the goal of this present investigation is to study  
the ovulation time in primiparous laboratory rats through the  
counting the number of graafian follicles present in the ovary  
at the time of oestrus, determining the percentage of these  
follicles that eventually ovulate, determining the 'spread' of  
ovulation during the oestrus period.  
Rats which have been adapted to tropical conditions are  
generally considered to have a regular oestrous cycle. Basic  
information concerning the spread and timing of ovulation and  
the optimum time of mating is needed to obtain maximum  
fertility both in natural and in reproductive research purposes.  
Information relative to such needs are lacking in Nigerian  
laboratories. Accurate detection of ovulation and its correct  
timing are, two prerequisites for the successful use of  
laboratory rats as reproductive research models. Behavioural  
observations measuring ovulatory manifestations and adequate  
Materials and Methods  
Animals  
For this study, 50 albino laboratory female rats weighing  
between 110g - 185g and some males, bred in Theriogenology  
laboratory of the Faculty of Veterinary Medicine, Ahmadu  
Bello University, Zaria, were used. Selected female rats must  
have whelped. Approval for this study was sought and gotten  
*
Corresponding author: Joy Iyojo Itodo, Department of Animal Science, Faculty of Agriculture Federal University of Lafia, Nasarawa,  
Nigeria. Email: iyojojoy@gmail.com  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 68-72. https://doi.org/10.47277/JIRB/8(4)/68  
from the Ahmadu Bello University Committee for animal use  
prevented the rats from struggling and it exposed the  
vaginal opening for the insertion of the swab.  
and care. Rats were housed separately and used for the  
detection of oestrus in the females. All rats were kept in plastic  
shoes-box cages covered with wire mesh tops, fitted with  
drinkers. Each cage was bedded with clean wood shavings. The  
work was carried out in Theriogenology laboratory of the  
Faculty of Veterinary Medicine, Ahmadu Bello University  
Zaria.  
For Rats Dissection and harvest of the ovaries, the rats  
were caught in the palm of the left hand with the thumb  
resting between the forelimbs and under the jaw to  
prevent bites, then a chunk of cotton wool soaked in ether  
was placed over the nostrils and mouth to prevent  
breathing of air until anaesthesia was achieved.  
Feeding  
The rats were fed with homemade pellets prepared with  
commercial chicken feed produced by feed masters company  
limited, composed of protein (4%), fats (2%) calcium (1%)  
phosphorus (0.4%) lysine (0.55%) methionine (0.25) with net  
energy of 2500 cal. /kg, palm oil as a source of vitamin A. The  
feed components were thoroughly mixed together and made  
into paste by adding water, and moulded into pellets. The  
pellets were dried in an electric oven at a temperature of 150  
Confirmation of Ovulation  
At the end of each group time interval, individuals were  
anesthetized. The ovaries were removed and pooled together  
for each time group. The left ovaries were kept separately from  
the right. Harvested ovaries cleaned of fat and other tissues  
were Fixed in Bouins solution for twenty four hours and then  
transferred to 70% alcohol. Ovaries were embedded, sectioned  
and stained for histological examination. The ovaries were  
serially sectioned at 5µm thick and five to six sections of each  
ovary picked at regular intervals in the series was placed, in  
sequence, on a slide for examination (6).  
°C. Dried pellets and water were given to the rats ad libitum  
and continued till the end of study. Drinking water was  
provided in sipper bottles suspended with the feed on the wire  
mesh. Other materials used are; recording materials, cotton  
buds, physiological saline, cotton wool, diethyl ether, slides,  
microscope, specimen bottles, dissecting kit, masking tape for  
sticking labels on cages, weighing scale.  
Analysis of ovulation rate  
The graafian follicles and corpora lutea were counted in the  
sections from each ovary and pooled, for left and right per time  
group of rats. For each pool, ovulation rate was calculated as:  
Experimental Groups and Behaviour  
The female rats were divided into 10 groups (n=5), each group  
corresponding to one of one to 10 equal intervals of the oestrus  
period, which is approximately one hour. For all groups,  
oestrus was detected in each female by the use of males. The  
first time the female stands to be mounted is the onset of  
oestrus.  
×
ퟏퟎퟎ  
풂 + 풃  
where a is the total number of corpora lutea counted per group  
and b is the total number of Graafian follicles still present in  
the same ovaries.  
Detection of Oestrus  
Graphical Representation of Data  
From personal communication, oestrus is known in albino  
rats, in the Theriogenology Laboratory where this work was  
carried to occur between 4:00pm and early hours of the  
morning. Therefore, male rats were introduced into the female  
cages shortly before 4:00pm daily and observed for behaviour  
and mating. Females rats pursued vigorously and standing to be  
mounted by the males were taken to be in oestrus. The time  
when females being pursued stood to be mounted was recorded.  
Groups of females were anaesthetized with diethyl ether  
inhalation at the end of the groups time interval, which was one  
hour, two hours, three hours and 10 hours for the respective  
groups.  
Proportionate number of ova released per group of rats or  
per time was plotted. This showed the relationship between the  
number of ova released and the time of their release during the  
oestrus period. The peak of the curve now represented the best  
time of ovulation. Time from the onset of oestrus  
corresponding to the peak of the curve will be recommended  
for use in the determination of ovulation time in albino rats.  
Results  
Oestrus period or heat was observed in all the fifty rats used  
in this study as the "female" standing to be mounted by the  
male. It was observed that the vagina at this time is wide open,  
pinkish and moist. Prior to standing for mounting, there is  
usually a period of vigorous running activity during which the  
male “disturbs” the female and the female resists mating. The  
period was considered the transition period from proestrus to  
oestrus in this study. This activity starts at different times for  
different female individuals but rarely before 1600 hours in the  
laboratory where this work was done.  
Vaginal Cytology  
Rats were restrained and cotton buds wet with normal  
saline was gently inserted into the vagina until it reached the  
anterior. Then, the bud was rolled once in the vagina for a swab  
and removed to prevent hyperstimulation which can lead to  
pseudo-pregnancy. The swab was placed on a clean microscope  
slide and rolled to achieve a smear. The smear was allowed to  
dry, dried slides were observed under the microscope for the  
cell types (5).  
From the longer time groups of 5 to 10 hours, the oestrus  
activity was observed not to last longer than 4 to 5 hours for  
each individual. From the time the females stands to be  
mounted for the first time, running activity reduces or stops as  
she remains to be severally mounted by the same male, or  
several others until intromission and ejaculation is achieved.  
There are usually several mountings before intromission and  
ejaculation is achieved.  
Restraints during Handling of Rats  
For vaginal smear collection, rats were placed on a  
table with the palm of the left hand placed over the  
dorsum, with a slight pressure, the thumb and forefinger  
were a used to pull the base of the tail backwards, this  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 68-72. https://doi.org/10.47277/JIRB/8(4)/68  
ovulation) and one without luteal phase (spontaneous  
Vaginal Cytology  
Vaginal smears taken at the time of first intromission  
presented only cornified superficial cells in all individuals. This  
cellular constitution did not change in any group of individuals  
when it was taken again at the end of the group's time period.  
ovulation) when the rats receive some certain stimuli.  
Nalbandov (2, 9) also reported that in spontaneous ovulators,  
LH is cyclic, independent of copulation but provoked by the  
interplay of the neuroendocrine system.  
Induced ovulation, on the other hand, occurs only when the  
cervix or part of the vagina is appropriately stimulated (10, 11).  
In this case, our oestrus rats that did not present functional  
structures could be grouped under the spontaneous ovulators  
producing GFs, ovulating but not developing CL (12). But  
these groups of rats involve LH action; therefore, the inability  
to produce CL is not quite clear. If the reason for lack of CL  
development is inappropriate induction, at least, an ovulated  
follicle could have been observed. There is a need for a better  
understanding of the physiology of the ovarian cycle in these  
groups of rats.  
About 67% of the obviously active rats showed signs of  
activity only on one ovary. It is not clear whether it can also  
be attributed to incomplete induction as they all carried  
primary and growing follicles but no GFs on the affected  
ovaries. Furthermore, it is reported that the left ovary is more  
active in mammals than the right ovary (6), observation in this  
study cannot quite stand with that bias because while  
correctly, there were higher follicular, activities on the LOs  
Functional Structures of the Ovaries  
The data on Graafian follicles (GF) and corpora lutea (CL)  
counted from the sectioned ovaries of 50 rats are presented in  
Tables' 1 - 3 and Figure 1. Contrary to expectation, some  
individuals did not present any functional structures at all on  
sectioning. Only group one had all the members presenting  
functional structures with two only on the right ovaries. One  
member each in group 2, 6, 9 and 10, and two each from 3, 4,  
5
, 7 and 8, making a total of 14 rats out of 50, did not present  
any GF or CL in both right ovaries (RO) and left ovaries (LO).  
Out of 36 rats with apparent ovarian activity, 11 (30.6 %),  
1
3 (36 %) and 12 (33.3 %) were active only on the RO, LO and  
both ovaries respectively. However, out of the 36, only 26 (72  
) showed any signs of ovulation in their ovary sections with  
%
rates ranging from 33 % to 100 % in different groups: All the  
members of groups six and seven and a couple of individual  
members in groups 1, 2, 3, 4, 5, 8 and 10, making 15 individuals  
presented 100 % ovulations of all GFs developed. On the other  
hand, the entire number of rats in group nine and another couple  
of individuals from groups 1, 3, 4 and 5 making a total of nine  
rats presented zero ovulations of all the GFs observed. Both  
extremes included all categories of active rats.  
(
42:36), ovulatory activities, on the contrary were higher on  
the ROs (58 %:54 %).  
This work also has no explanations for complete  
ovulations in one quarter of the active rats.  
A total of 78 GFs (36 RO, 42 LO) with 47 (60 %; 23 RO  
and 24 LO) becoming CLs were obtained from a pool of all the  
rats. Ovulation rates pooled for active only on the ROs or LOs  
were 58 % (11 CLs of 19 GFs) and 54 % (13 CLs of 24 GFs)  
respectively. Of the 12 rats active on both ovaries, ovulation  
rates were spread as 0 %, 33 %, 43 % 50 %, 67 %, 80 % and  
Conclusion  
It can be concluded from this work that what triggers the  
"stance" for mounting in the albino laboratory' rat is the  
mechanism behind the ovulation process. It can also be  
concluded that any follicle that did not ovulate at the time of  
onset of mounting may not ovulate again. Therefore, we  
conclude that the ovulation time in the laboratory albino rat in  
our laboratory, and probably elsewhere, is the beginning of  
oestrus, peaking maximally at the sixth and seventh hour of  
oestrous. However, every observation needs validation in  
wider studies.  
1
00 % in 1, 2, 1, 3, 2, 1 and 2 rats respectively. Total average  
for ovulation rate in this study is 60 %.  
Discussion  
This study was aimed at pegging the ovulation time for  
rats in our local laboratory for greater usefulness as  
reproductive research models. Ovulation occurs within the  
oestrus period in most mammals (6) and has been reported to  
occur at 8-10 hours into oestrus in rats with a heat period of  
1
2-15 hours (2, 5). Though no data was collected on the heat  
period of the laboratory rat in this study, it appeared to be  
much shorter than 12 hours. This is in contrast with Paccola et  
al., (1) that found out that the ovulation was 12 hours with an  
average duration of estrous cycle ranges from 3.5 to 5.5 days.  
This substantiates the need for knowledge of the exact  
ovulation time in the laboratory rats used in our own  
environment. From the tables and the figure presented, it is  
clear that ovulation time is not pegged in the laboratory albino  
rat, at least in the laboratory where the work was carried out.  
It is more or less a spread from the onset of oestrus (7). From  
the high ovulation rate observed in the first hour (72%), it is  
possible that the rest of the groups could have ovulated in the  
first hour also.  
It is also confusing, that about 33% of the rats that  
participated in vigorous sexual activity and were confirmed to  
be in oestrus by vaginal smear examination could neither  
present CLs nor GFs . Well, Krinke (8) reported two types of  
cycle in the rats such as a cycle with a luteal phase (induced  
Figure 1. Ovulation rate in different groups of rats  
corresponding to different hours of oestrus periods  
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J Infertil Reprod Biol, 2020, Volume 8, Issue 4, Pages: 68-72. https://doi.org/10.47277/JIRB/8(4)/68  
Table 1: Numbers of Graafian Follicles and Corpora Lutea in the Ovary Pairs of Individual Rats, Groups of Rats, their Averages  
and Percentage Ovulations  
Groups  
RAT  
Left ovaries  
Ovulation  
Left ovaries  
Ovulation  
Corpora Lutea  
Graafian follicles  
(%)  
100  
67  
100  
0
50  
70  
0