Contributor: Prof. Jogen C Kalita, Animal Physiology and Biochemistry Lab, Department of Zoology, Gauhati University

1. ESTROUS CYCLE AND ITS
HORMONAL REGULATION
PROF. JOGEN C. KALITA
MSc (Gau), PhD (King’s College London)
Professor in-Charge
Animal Physiology & Biochemistry Lab.
Dept. of Zoology, Gauhati University,
Guwahati, Assam, India

2. After going through this PPT the students will be able to learn the following:
• Know about Estrous cycle and the cyclic nature of reproductive activities
in female mammals
• Different phases of the estrous cycle in mammals
• They will know about physiological changes happening internally inside
animals body can be assessed by simple external observation using
vaginal smears technique (without using any invasive/aggressive
methods)
• Know about the hormonal feedback that exists among hormones of
Hypothalamus, Pituitary and Ovaries for regulation of the cycle
• Applied value of the earned knowledge in the field, animal husbandry
and in case of farm animals (induced breeding etc.)
LEARNING OUTCOMES

3. DEFINITION OF ESTORUS CYCLE
& RELATED TERMINOLOGY
• A cycle is a repetition of the same event (s) at a definite interval of
time.
• The Estrous or Oestrous Cycle is the reproductive cycle occurring in
non-primate mammals.
• The repetition of morphological/physiological and behavioural changes
occurs in female mammals at a definite interval of time. This duration
of time interval differs in species.
• The Estrous cycle has four distinct phases, proestrus, estrus,
metestrus and diestrus.
• During their reproductive life female mammals exhibit two types of
reproductive cycles, Estrous cycle and Menstrual cycle. Human
female and primates have Menstrual Cycle. Unlike estrous cycle the
Menstrual Cycle has a distinct bleeding phase.

4. DEFINITION OF ESTORUS CYCLE
& RELATED TERMINOLOGY
• We should not confuse with the term "estrus“. Estrus is just a phase
of the Estrous Cycle. The word “estrus” means psychological heat
period (psychic heat) of receptivity of female towards male for
mating. Only during this estrus period of the cycle a female receives
a male for mounting.
• Estrous cycle starts after sexual maturity in females and are
interrupted by anestrous (absence of estrous) phases or by
pregnancies.

5. The word “estrus” was first used by Heape (1900). This is a Latin form of
the Greek word “oistros” which means “sexual season”, “gadfly”, “frenzy”,
“sting” or “madness”. Heape further named and defined the phases of the
mammalian estrous cycle into proestrus, metestrus, diestrus and anestrus.
 Proestrus is the preparatory stage for an animal coming into heat. Estrus
is the actual period of heat when females receive male for mounting.
 Metestrus which is a brief period characterized with decline of corpus
luteum functions in the absence of conception when the activities of
reproductive organs gradually subside,
 Diestrus which is a period of short rest during the breeding season, and
 Anestrus which a non-breeding period when reproductive organs are
quiescent.
HISTORICAL BACKGROUND

6.  Proestrus: The first stage in the estrous cycle immediately before estrus
characterized by development of both the endometrium and ovarian follicles.
 Estrus: The second stage in the estrous cycle immediately before metestrus
characterized by a receptivity to a male and to mating, often referred to as "heat" or
"in heat". Pheromones may also be secreted only at this stage of her cycle.
 Metestrus: The third stage in the estrous cycle immediately before diestrus
characterized by sexual inactivity and the formation of the corpus luteum.
 Diestrus: The last stage in the estrous cycle immediately before the next cycle
proestrus characterized by a functional corpus luteum and an increase in the blood
concentration of progesterone.
 Anestrus: Not a stage in the estrous cycle, but a prolonged period of sexual rest
where the reproductive system is quiescent (inactive).
STAGES OF THE ESTROUS CYCLE
The descriptions below refer to the "typical" estrous cycle
Proestrus - Estrus - Metestrus - Diestrus

7. DIFFERENCES BETWEEN
ESTROUS CYCLE AND MENSTRUAL CYCLE
Estrous cycles are named for the cyclic appearance of behavioral sexual
activity (estrus) that occurs in all mammals except for higher primates.
Menstrual cycles, which occur only in human female and primates, are
named for the regular appearance of menses due to the shedding of the
endometrial lining of the uterus.

8. ESTROUS CYCLE MENSTRUAL CYCLE
Estrous cycle occurs in Infra-primate female
mammals only, for example rats, mice, cat,
dog, cow, horse, pig etc.
Menstrual cycle occurs In human female
and primates
Estrous cycle has its four distinct phases,
proestrus, estrus, metestrus and diestrus,
where receptivity to males is limited to
estrus phase only.
Menstrual cycle has no such type of
proestrus, estrus, metestrus or diestrus
phases and female can mate with male
during the entire cycle period.
Estrous cycle is normally of short duration
starting from 4-days
Menstrual cycle duration is always of about
a months (28±2 days)
Estrous cycle has no distinct bleeding
phase
Menstrual cycle has a distinct bleeding
phase towards the end of each cycle
DIFFERENCES BETWEEN
ESTROUS CYCLE AND MENSTRUAL CYCLE

9. ESTROUS CYCLE
TYPES
In different mammals during their reproductive life, Estrous cycles vary between
species
 Polyestrous Animals: Estrous cycles occurring throughout the time of the year
(e.g, cattle, pigs, mice, rats). They can become pregnant without regard to the
season of the year.
 Seasonally Polyestrous Animals: Animals that have multiple estrous cycles only
during certain periods of the year (e.g, horses, sheep, goats, deer, cats). They
are classified as “short day breeders” when they present estrous cycles during
autumn when day length is decreasing (goats and ewes) and “long day breeders”
(mares and queens) when they show estrous mainly during spring, when day
length increases.
 Monestrous Animals: Animals that have one estrous cycle per year (e.g, dogs,
wolves, foxes, and bear)

10. CHANGES IN REPRODUCTIVE PHYSIOLOGY
DURING ESTROUS CYCLE
Female mammals always exhibit different kind of
behavioural, as well as morphological, histological, cytological, and physiological changes in
the reproductive tract and also hormonal changes during each estrous cycle as stated below
PROESTRUS
 Follicles in ovaries start developing further
 Estrogen, the main female sex hormones of
ovary increases in circulation
 Estrogen start acting on its target, uterus and
vascularity of the female reproductive tract
increases
 Inside uterus endometrial thickness and
glands of endometrium begin to grow
 Estrogen level becomes the highest (levels
peak)

11. ESTRUS
 During this time of the cycle female allows
male to mate (mounting)
 Estrogen in circulation slowly decreases
 LH from anterior Pituitary level increases to
peak (surge occurs)
 Ovulation occurs after few hours of surge of
LH or during the surge of LH
 Uterine motility becomes high with
contractions moving toward the oviduct
 Sperm transport is optimal during this time if
mating occurs and cervical mucus volume
increases

12. METESTRUS
 Circulating Estrogen level becomes low
 Corpus hemorrhagicum present
 Ovulation may occur in some animals
 FSH increases, triggering growth of
follicles
 Uterus
 contractions subside
 endometrial glands continue to
grow and become coiled

13. DIESTRUS
 Progesterone secreted from ovaries
become higher in circulation
 FSH level becomes low
 Uterus
• Secrets fluid but the volume of fluid
decreases over time
• Contraction stop
• Corpus luteum regresses at the end
of this period if female is not
pregnant

14. STUDY OF ESTROUS CYCLE USING
VAGINAL SMEARS IN RATS AND MICE
BACKGROUND
 The estrous cycle in rats and mice averages 4–5 days and is a repetitive but
dynamic process whereby different cell types appear and recede in waves
throughout the cycle, reflecting changes in the levels of estradiol and
progesterone secreted by the ovarian follicles and further changes occurring in
the uterus, mainly endometrium, cervices, and vagina.
 STOCARD & PAPANICOLAOU (19I7) dealt with the maturation process in the
Graafian follicles using the smear method with vaginal fluid in the guinea pig.
 ALLEN (1923) and HISAW (1947) put forwarded a theory on the oestrous cycle in
the Muridae (largest family of rodents). Estrous cycles are characterized by
morphological changes in ovaries, the uterus and the vagina which occur during
different phases in the cycle (Goldman et al., 2007).
 Vaginal smear method remains as the gold standard till date to study estrous cycle
in commonly used experimental animals.

15. VAGINAL SMEAR PROCEDURE
Estrous cycle can be studied by collecting vaginal smears with a sterile cotton
plugs putting in to the vagina or by a dropper. We in our Lab used a sterilized
stainless steel wire loop having a diameter of about 3.5 mm was dipped in
distilled water to create a diaphragm of water inside the loop. One drop of
distilled water was added on the middle of a clean microscopic slide. The loop
was then inserted into the vagina and rotated gently 2-3 times to collect the
vaginal cells and finally placed onto the water drop and spread softly to make a
smear. The slide was then allowed to air dry, fixed in methanol and stained for
10-15 min in Giemsa stain and observed under normal microscope. Four
distinct cell associations of vaginal cytology appear at four different stages of
the cycle.

16. Animal Proestrus Estrus Metestrus Diaestrus
Mice(4-6 days) 24hrs 24hrs 12 60±4
Rat (4-5 days) 12hrs 12hrs 21hrs 57±1hrs
Estrus cycle length in Rodents (Mice & Rat),
Cora et. al., 2015

18. Fig: Appearance of cytological changes in four phases of estrous cycle in mouse
The different phases of oestrous cycle are usually identified according to cell
types observed in vaginal smears
VAGINAL CYTOLOGICAL CHANGES DURING
OESTROUS CYCLE & IDENTIFICATION OF THE PHASES

19. Phase of the cycle Cell types
Proestrous Large rounded nucleated epithelial cells with distinct plasma
membrane (PM) and clear cytoplasm.
Estrus Entirely of cornified cells, in high numbers and usually forming
clumps and sheets. Cells are ells with granulated cytoplasm.
Cells with no distinct boundaries (PM) and nucleus.
Metestrous Large numbers of leucocytes and smaller numbers of mostly
large, non-granular and non-nucleated epithelial cells.
Diestrous Mainly of leucocytes but sometimes with quite variable
numbers of epithelial and small cornified cells
Table: Cytological changes during
4 different phases of estrous cycle

20. Estrous cycle is a hormonally controlled cycle of activity of the reproductive organs in
female mammals. The follicular stage (growth of the Graafian follicles, thickening of
the lining of the uterus, secretion of oestrogen, and ovulation, is succeeded by the
luteal phase (formation of the corpus luteum and secretion of progesterone), followed
by regression and a return to the first stage. The first half of the cycle is also termed
as Follicular phase or Estrogen phase due to estrogen dominance and the second half
of the cycle is called the Luteal phase, as corpus Luteum forms during this time or as
Secretary phase or Progesterone phase.
There are four major hormones involved in the oestrous cycle. These hormones
include
•Follicle-stimulating hormone
•Luteinizing hormone
•Estrogen
•Progesterone
HORMONAL REGULATION OF THE ESTROUS CYCLE

21. Fig: Relationship between circulating reproductive hormones, reproductive cycle stage, and
vaginal cytological outcomes through different phases estrous cycle in female mouse.
(Jessica G. et. al., 2018)

22. The cycle is regulated by the hypothalamic-pituitary-gonadal axis, which produces
hormones that dictate reproductive events. The reproductive axis is composed of the
hypothalamus, pituitary, and the ovary
HORMONAL FEEDBACK DURING ESTROUS CYCLE
Fig: Hypothalamic Pituitary gonadal Axis,
the interaction of the structures and
hormones involved within the oestrous cycle

23. The sequence of hormonal release essentially begins with the synthesis and release
of GnRH from the hypothalamus. This polypeptide hormone is transported to the
anterior pituitary through a highly specialized capillary network called the
hypothalamo-hypophyseal portal system. GnRH functions to stimulate the anterior
pituitary to produce and release FSH and LH. FSH and LH are transported through
systemic blood circulation to the ovaries, where they initiate a series of morphological
changes in the ovaries (ovarian folliculogenesis) that lead to ovulation (and
pregnancy if fertilization occurs). The primary hormones produced by the ovary are
Estrogen and Progesterone. These hormones are transported by the blood stream to
"target" tissues (mainly uterus and breast) to cause a development. As the follicle
grows, more estrogen is produced. As increasing amounts of estrogen are released
into the blood stream and travel to the anterior pituitary, it acts in a positive feedback
fashion, stimulating pulsatile LH release. The uterus found in the pelvic cavity also
contributes to reproductive control, as it produces prostaglandin F2a(PGF2a).

24.  Changes occurring in the mouse estrous cycle are evident in the animal’s
physiology and anatomy. The cells lining the vagina of the female rat respond to
the levels of circulating hormones and can provide a valuable marker of the
stage of preparation of the ovary and uterus for mating and for ovulation.
 Vaginal smears taken on consecutive days over a period of time can provide
detailed information on the oestrous cycle. The normal oestrous cycle in the rat
usually follows a 4-day pattern and the varying characteristics of the cells in the
smear allow the days of the cycle to be classified relative to the predicted time of
ovulation. Ovulation occurs at approximately midnight after the pro-oestrous
stage, when the females become receptive to the male.
IMPORTANCE OF STUDY OF ESTROUS IN ANIMALS

25.  The stages of the vaginal cell cycle will normally correlate with changes
in the female reproductive organs e.g. ovary and uterus. This
knowledge can be used in many lab experiments to study reproductive
performances of animals under different situation and for testing drugs
etc. and in the field of induced breeding technology.
 In farm animals and animal husbandry the detection of estrus and
ovulation time is very important for farmers to specifically apply mating
at the specific time.
IMPORTANCE OF STUDY OF ESTROUS IN ANIMALS

26. SUGGESTED READINGS
Essential Reproduction (Essentials): Amazon.co.uk: Martin H. Johnson,
8th Editon
Knobi and Neil’s Physiology of Reproduction, Vol. 2, 4th Edition
The murine female intestinal microbiota does not shift throughout the
estrous cycle
Jessica G. et al., Published: July 16, 2018
https://doi.org/10.1371/journal.pone.0200729

27. THANK
YOU