Screening of ANTIFERTILITY AGENTS and APHRODISIACS

EVALUTION AND SCREENING
MODELS FOR ANTIFERTILITY
AGENTS
Presented by : Tailor Himani Rajubhai
Department of Pharmacology
L. M. College Of Pharmacy

●Anti Fertility drugs
• Antifertility drugs are chemical substances which suppress the
action of hormones that promote pregnancy.
● These drugs actually reduce the chances of pregnancy and act
as a protection. Antifertility drugs are made up of derivatives of
synthetic progesterone or a combination of derivatives of
estrogen and progesterone .
● • These are also known as contraceptive agents.
● • Contraception is the method of preventing normal process of
ovulation, fertilization and ovum implantation , nothing but
pregnancy.
● • These are classified into two types.
● (1) Female contraceptive agents.
● (2) Male contraceptive agents .

●Mechanism of Action
● Hormonal contraceptives interfere with fertility in many ways; the
relative importance depends on the type of method as:
● 1. Inhibition of Gonadotropin release from pituitary by
reinforcement of normal feedback inhibition by progestin, reducing
frequency of LH secretory pulses (an optimum pulse frequency is
required for triggering ovulation) while the estrogen primarily reduces
FSH secretion. (minipill and progestin-only)
● 2. Thick cervical mucus secretion hostile to sperm penetration is
evoked by progestin action.
● 3. Even if ovulation and fertilization occur, the blastocyst may fail to
implant because endometrium is either hyperproliferative or
hypersecretory .(minipill & post coital)
● 4. Uterine and tubal contractions may be modified to disfavour
fertilization. This action contributes to the efficacy of minipills and
postcoital pill.

●
Ovarian changes during menstrual
cycle

●Physiological changes during
pregnancy
● If ovulating normally, an egg, or ovum emerges from one or other of
your ovaries, leaving behind a structure called the corpus luteum. This
structure produces large amounts of progesterone and estrogen,
hormones that help prepare your uterus for implantation of a fertilized
egg.
● If the egg is not fertilized, the corpus luteum degenerates, causing
progesterone and estrogen levels to drop, and menstruation to begin. If
the ovum is fertilized, on the other hand, the corpus luteum remains
intact and continues to maintain the hormone levels you need to keep
your uterus baby-friendly.

Types of contraceptive preparations
(female contraceptives)
A) Oral
● 1. Combined pill
● 2. Phased regimens
● 3. Mini pill (progestin only)
● 4. Postcoital (Emergency)
B) Injectables
● 1. Long acting progestin only
● 2. Long acting progestin + Long acting Oestrogen
C) Implants
● 1. Biodegradable implants
● 2. Non biodegradable implants

MALE CONTRACEPTIVE :
• The only way to suppress male fertility by drugs is to inhibit
spermatogenesis. Though considerable effort has been made in this
direction and effective drugs have been found, no satisfactory/ acceptable
solution is yet tangible.
• Drugs and approaches tried are—
• 1. Antiandrogens : Act by direct action on testes; cause unacceptable loss of
libido.
• 2. Estrogens and progestins : Act by suppressing Genital nerve
stimulation— cause unacceptable feminization.
• 3. Androgens : They inhibit Genital nerve stimulaton but have poor efficacy;
combination with progestin is more efficacious, preserves libido, but is still
not reliable.
• 4. Superactive Gn RH analogues : They inhibit Gn release after continued
action; also inhibit testosterone secretion— produce impotence, loss of
libido.
• 5. Cytotoxic drugs : Cadmium, nitrofurans and indoles suppress
spermatogenesis, but are toxic, often produce irreversible action.

METHODS FOR FEMALES
A) Methods Antiovulatory Activity
● 1. HCG induced ovulation in rats.
● 2. Cupric acetate induced ovulation in rabbits.
B) Progestational activity
● In vivo methods :
● 1. Pregnancy maintenance test.
● 2. Proliferation of uterine endometrium in oestrogen primed
rabbits (Clauberg Mcphail test )
● In vitro methods :
● 1. Progesterone receptor binding assay.

C) Oestrogenic activity
● In vivo methods :
● 1. Vaginal opening
● 2. Assay for water uptake
● 3. 4-day uterine weight assay
● 4. Vaginal cornification
● 5. Chick oviduct method
● In vitro methods :
● 1. Oestrogenic receptor binding assay
● 2. Potency assay

●Methods for halting reproduction in
females :
• Female antifertility agents might be acting through
following mechanisms :
1. Inhibition of ovulation.
2. Prevention of fertilization.
3. Interference with transport of ova from oviduct to
endometrium of the uterus .
4. Interference with the implantation of fertilized
ovum.
5. Distraction of early implanted embryo.

A) Tests used for screening :
Anti ovulatory activity
● 1) HCG – Induced ovulation in rats :
Rationale :
• Immature female albino rats do not ovulate spontaneously
and do not show cyclic changes of the vaginal epithelium .
• Priming with HCG induces follicular maturation ,
followed by spontaneous ovulation 2 days later .
• Injection of anti ovulatory drugs, prior to induction
procedure will prevent ovulation .
• This principle is used for the screening of anti-ovulatory
agents

Procedure :
• Immature female albino rats 24-26 days of age are used for
the experiment .
• The animals are treated with various test drugs in a different
dose levels .
• After the administration of the test drug ,HCG is given
exogenously for ovulation.
• After 2 days ,animals are sacrificed ,ovaries are dissected out
,preserved in 10% buffered formalin and subjected to
histopathological evaluation .
• The results are compared with the control group.

2) Cupric acetate-induced ovulation
in rabbits
Rationale :
The rabbits are reflex ovulators .They ovulate within a few hours
after mating or after mechanical stimulation of vagina or
sometimes even presence of males ,or administration of certain
chemicals like cupric acetate .
• The rabbit ovulates within a few hours after injection of cupric
acetate (0.3mg/kg i.v of 1 %cupric acetate in 0.9 saline ) .
• Injection of anti ovulatory drugs ,24 hours before the induction
procedure prevents ovulation.

Procedure
• Sexually mature female rabbits, weighing 3-4 kg are used for the
study. Animals are kept in isolation for atleast 21 days to ensure
,they are not pregnant and to prevent the induction of ovulation
by mating .
• They are treated with test drug and 24 hours later cupric
acetate is given .The rabbits are sacrificed and ovaries are
examined 18-24 hrs. later .
• The total number of ovulation points on both ovaries are
recorded for each animal .
• Then the ovaries and uterus are excised out and preserved in 10%
buffered formalin and subjected to histopathological evaluation .

B) Oestrogenic activity
• A primary therapeutic use of Oestrogen is in contraception .The
rationale for these preparations is that excess exogenous
Oestrogen inhibits FSH & LH , thus prevents ovulation.
● In vivo Methods :
1) vaginal opening :
• This assay is based on the principle that vaginal opening occurs
in immature female albino mice & rats by treating with
Oestrogenic compounds . The sign of complete vaginal opening is
observed as a sign of Oestrogenic activity.

Procedure :
• Immature female animals ( 18 day old mice , 21 day old mice
rats) are used for the study.
● The test & standard
drugs are administered to
the animals intramuscularly
in cotton seed oil .
Evaluation: The time of complete vaginal opening can be observed
as a sign of Oestrogenic activity.

●Four-day uterine weight assay
Principle
● This assay is based on the observation that estrogens cause an increase in protein
synthesis and thus bring about an increase in uterine weight. A peak in uterine
weight is observed about 40 hrs.
Procedure
● Immature or ovariectomized mice or rats can be used.
● Injections are given intramuscularly in cottonseed oil for 3 consecutive days.
● On the fourth day animals are killed by cervical fracture, the uteri are rapidly
excised, and the uterine contents are gently squeezed out (results are unreliable if the
uterine contents are not removed).
● The uteri are weighed immediately in the wet state.
● The uteri may be dehydrated in an oven at 100° C for 24 hr and re weighed to
obtain the dry weight increase.
● Plotting log dose against the wet weight produces a sigmoid curve, and the ED5o
can be determined for comparison of the test compound with estradiol

Vaginal cornification
Rationale :
• This assay is based on the fact that rats and mice exhibit a cyclical
ovulation with associated changes in the secretion of hormones
,this lead to the changes in the vaginal epithelial cells.
The oestrus cycle is classified into
1. Proestrus.
2. Estrus.
3. Metestrus.
4. Diestrus .
• Drugs with Oestrogenic activity change the animal into estrus
stage.

Stage of estrus cycle in rats
• The estrus cycle is a cascade of hormonal and behavioral events which are highly
synchronized and repetitive. The short and precise estrus cycle of the laboratory rats
has been a useful model for reproductive studies .
• The estrous cycle of rat is usually completed in 4-5 days as :
1. Proestrus : is beginning of new cycle ,follicles start maturing under the influence of
Gonadotropic Hormone and the estrogen secretion start increasing , smear is
characterized by by nucleated epithelial cells ,stage lasts for about 12 hrs.
2. Estrus : uterus is enlarged and extended due to fluid accumulation , oestrogen
secreation is at its peak , smear shows squamous cornified cells. Estrus means period
of heat or sexual receptivity. (12 hrs).
3. Metestrus : ovary contains corpora lutea , secreting progesterone , indicated by
mixture of leukocytes & cornified epithelial cells indicating post ovulatory stage.(21 hrs)
4. Diestrus : corpora lutea regress & declining secretion of oestrogen &
progesterone causes regression of the uterus, smear shows only leukocytes . (57 hrs)

●Apperance of external genetelia

●Preparation of vaginal smears

● Evaluation
● A proestrus smear will have many
epithelial cells with granular cytoplasm,
indicating a rapidly growing vaginal
epithelium and the preovulatory stage .
● A positive estrus smear is one in which
only large, irregular cornified cells are
seen, indicating maximum growth of the
vaginal mucosa.
● A metestrus smear will have many
cornified cells, but also some
leukocytes and epithelial cells, indicating
the postovulatory stage and desquamation
of the vaginal mucosa.
● A diestrus smear will show few
epithelial cells, mucus, and few
leukocytes, indicating a quiescent uterus
and resting vaginal epithelium.

●Chick oviduct method
Rationale :
• The weight of oviduct of young chicken is increased, dose –
dependently by natural and synthetic Estrogen .This principle is
used for the screening Estrogenic compounds.
Procedure :
• 7 days old pullet chicks are injected subcutaneously , twice daily
with solutions of the test compound in various doses for 6 days .
• Doses between 0.020 & 0.5 μg ,17 β estradiol per animal serve
as standard . • 6-10 chicks are used for each dosage group.
• On the day after the last injection , the animals are sacrificed and
weight of the body and oviduct is determined .

●In vivo methods
● Estrogen receptor binding assay = uses the
principle of competitive binding of labeled and
unlabeled estrogen on the estrogenic receptors.
● Estrogenic compounds displace the labeled
estrogen in a concentration dependent manner
from the estrogen receptor.

Procedure:
● Uterus removed from Female albino mice (18 days old)
● Homogenized at 0 C OF Tris sucrose buffer in a conical homogenizer.
● Human endometrium from menopausal women is frozen within two
hour of hysterectomy.
● Stored in liquid nitrogen until use
● Frozen endometrium homogenized in Tris- sucrose buffer.
● Homogenates are centrifuged for 1 h Determination of specific
binding in mouse uterus cytosol as a function of steroid conc.
Incubation time and temp.
● Triplicate aliquots of 125 ml of cytosol are incubated with 5 or 25 nm
labeled steroid either for 2 or 24 h at 0 C for 2 or 5 hours at 25 C

●C) Progestational activity
● In vivo methods
Proliferation of uterine endometrium in
oestrogen primed rabbits (Clauberg Mcphail
test)
Principle :
• Female rabbits weighing between 800-1000g
are primed with estradiol and followed by the
administration of progestational compound
leading to the proliferation of the endometrium
and converted into secretory phase .

Procedure :
• Female rabbits weighing 800- 1000g are primed with injection of
estradiol 0.5ug/ml in aqueous solution daily .on day 7 treatment
is begun .
• The total dose is given in 5 equally divided fraction daily over
5 days .
• 24hrs after the last injection , animals are killed and uteri are
dissected out and frozen sections of segment of middle portion of
one horn is prepared and examined for histological interpretation .
• For interpretation of progestational proliferation of endometrium ,
beginning of glandular development may be graded 1 &
endometrium consisting only of glandular tissue may be graded 4

SCREENING MODELS FOR APHRODISIAC
AGENTS
33

Introduction
•Aphrodisiac is the word derived from “Aphrodite”, the Greek
goddess of sexual, love and beauty.
•Definition :An aphrodisiac is defined as an agent (food or drug)
that arouses(enhances) sexual desire(pleasure).
•The aphrodisiac drugs act by altering the level of specific
neurotransmitters or specific sex hormones into the body.
•Most of the aphrodisiacs agent acts by altering the testosterone and
progesterone concentration in the body.
34

MECHANISM
•These drugs act by enhancing the sex organ sensation
and performance. They improve the blood flow to the
male sex organs, thus improving the male libido. A
similar response in women may also produce an
increased sexual stimulation.
35

•mechanism which causes penile erection is through
cyclic adenosine monophosphate pathway
(cAMP).
Corporal smooth muscle relaxation is mediated via
cAMP.
-The activated membrane-bound adenylyl cyclase,
which generates cAMP, it activates protein kinase A
and to a lesser extent, protein kinase G.
-Prostagladin E1 also increases the intracellular
concentrations of cAMP in the corpus cavernosum
smooth muscle cells.
-The generation of cAMP activates the Ca2+ pump
and consequently, the level of free cytoplasmic Ca2+
is reduced, resulting in smooth muscle relaxation.
-Similarly, the protein kinase activates the cell-
membrane Ca2+ pump, leading to a decreased
sarcoplasmic Ca2+ concentration which induces a
loss of contractile tone of the penile smooth muscle
and increase of blood flow in the cavernous body
resulting in erection.
36

Guidelines to follow during
experiment
• Males were kept individually but females were kept in groups.
• Training of each male for 15 min at a time was performed until sexual
behavior was elicited and when the behavior was noticed, males were exposed to
receptive females (1 male with 5 females);
• Repeated training to overcome the lack of sexual response in the presence of
observers;
• The study was conducted in a silent room under dim red light;
• Any jerking movement of the mating area was avoided to enable the rats to
chase each other;
• Cleaning of the mating area was performed after each trial, since the urine
trails left by one rat might alter the sexual behavior of the next rat.
37

Aphrodisiac agents
•Sildenafil citrate
•Arginine
•Testosterone
•Crocin
•Phenethylamine
Amphetamine
Methamphetamine
38

●EVALUATOIN OF APHRODISIAC AGENTS :
The therapeutic value , efficacy and toxicity of drug may be evaluated in animals
experimentally ,followed by clinical trial.
• Both in- vivo & in - vitro animal models are employed to assess aphrodisiac
activity in experimental animal (rat, mice, guinea pig etc.)
• Some models can explain as ….
1) Physical Models :
Mating behavior test
-Mount frequency
-Mount latency
-Intromission frequency
-Intromission latency
-Post ejaculatory interval
-Copulatory rate
-Index of libido
-Computed male sexual behaviour parameter

2) Test for libido
3) Test for potency
4) Penile microcirculation study
5) ICP ( intra cavernous pressure) study
6) Orientation behavior
7) Determination of hesitation time & attraction towards female
8) Pendiculation study
9)Biochemical study :
-Determination of testicular and serum cholesterol (Chod PAP Method)
-Hormonal determination assay
10) Assay for neuronic NO Synthase(NOS) and androgen receptor protein
11) In – vitro NO Release
12) Effect on sexual organ weight & histological studies.
13) In – vitro Sperm preservation
14)In-vivo sperm count
15) Fructose content in seminal vesicles

●
A)Physical or Behavioral method(in vivo)
Mating behavior test
● Experimental conditions
Animal – Male Albino Rat (shows brisk sexual activity)
Red dim light in lab during experiment
Animals are trained prior to experiment
● Procedure: Healthy and sexually experienced male rats(200-300 g)
are divided into 3 groups.
GROUPS DRUG
I Distilled water(10 ml/kg)
II Test drug/agent(for 21
days)
III Standard
drug(sildenafil)(5 mg/kg)41

•Standard drug is administered 1 hour prior to the experiment
•Then all the animals are exposed to dim light (1 w fluorescent tube) at a stipulated
time of testing daily for 6 days prior to the test.
•Female rats are treated with
- 100µg/kg ethinylestradiol suspension (before 14 hrs ;orally)
-1mg/kg progesterone (before 6hrs;s.c)
•The treated female rats are introduced into the cage of male animal with the ratio
(1:1/1:2)
•Then next step is to observe and evaluate the mating behavior.
.
42

Evaluation : -
To observe mating series.
- Occurrences of events & phases of mating should be recorded .
- The parameters of male sexual behavior that should be monitored.
- If male rat failed to sexual interest, then modified the test like if
any female rat do not shows receptivity should be replaced by
another ‘warmed’ female

EVALUATION PARAMETERS
a.Mount frequency
-Mounting is defined as the climbing of one animal by another usually
from the posterior end with the intention of introducing one organ into
another.
-(MF) is therefore the number of mounts without intromission from the
time of introduction of the female until ejaculation.
b. Intromission frequency
-Intromission is the introduction of one organ or parts into another. e.g.
the penis into the vagina.
-Intromission Frequency (IF) is therefore defined as the number of
intromissions from the time of introduction of the female until
ejaculation.
44

c. Mount latency
-Mount latency (ML) is defined as the time interval between the introduction of
the female and the first mount by the male.
d. Intromission latency
-Intromission latency (IL) is the time interval from the time of introduction of the
female to the first intromission by the male.
e. Ejaculatory latency
-Ejaculation is the act of ejecting semen brought about by a reflex action that occurs
as the result of sexual stimulation.
-Ejaculatory latency (EL) is defined as the time interval between the first
intromission and ejaculation.
-This is usually characterized by longer, deeper pelvic thrusting and slow dismount
followed by a period of inactivity or reduced activity.
45

f. Post-ejaculatory interval
-Post-ejaculatory interval (PEI) is the time interval between ejaculation
and the first intromission of the following series.
g. Copulatory rate
Corpulatory rate=
no. of mounts + no. of intromission
Time from first mount till ejaculation
h. Index of libido
% index libido =
number mated
number paired
x 100
46

Computed male sexual behavior parameters
Using the above parameters of sexual behavior, the following can thus be computed:
● The parameters can be counted as:
I. % Mounted =
Number mounted
Number paired
× 100
II. % Intromitted =
Number of intromissions
Number paired
x 100
III. Intromission ratio =
Number of Intromission
Number of mounts + Number of intromissions
x100
IV. % Ejaculated =
Number of ejaculations
Number paired
x100
V. Copulatory efficiency =
Number of intromissions
Number of mounts
x 100
VI. Intercopulatory efficiency = Average time between intromissions
47

•Test for libido
-Sexually experienced male albino rats should be kept singly in separate cages
during the experiment.
-The female rats should be made receptive by hormonal treatment and all the
animals should be accustomed(familiar) to the testing condition as previously
presented in mating behavior test.
-The penis should be exposed by retracting the sheath and apply
5% xylocaine ointment 30, 15 and 5 min before starting observations.
Evaluation
-No. of mounting should be noted.
-The animal should also be observed
for intromission and ejaculation.
48

Test for potency
● The male animals are kept singly.
● The aphrodisiacs are administered before half or one hour of the
experiment.
● On the 8th day test for penile reflexes would be carried out by putting the
animal on its back in a glass cylinder with partial restrain.
● The perpetual sheath would be pushed behind the glans for 15 min to
stimulate genital reflexes.
● Total penile reflexes are obtained as sum of erections(E), quick flips(QF)
and long flips(LF).
i.e. E + QF + LF is obtained
49

Evaluation
Statistically significant increase in
the frequency of penile reflexes
suggests with aphrodisiac potential.
A= immediately after the retraction
of the penile sheath (no evidence of
erection),
B= E3 (cup),
C=long flip.

Penile microcirculation
study :
• Laser Doppler flow meter may
used for determine penile micro
circulations
• I.V. anesthesia of phenobarbital
sodium would given (dose 50
mg/kg body weight) to rat .
• Penile sheath would retracted
manually, after 10 min of
adaptation the Laser Doppler
Flow detection probe would be
positioned in a holder close (2-3
mm) to the dorsal side of penis.
• Average flow units (flux) within
10 min of the test would be
calculated.

●Intracavenous pressure (ICP) study
-Followed by the incision on the penile skin of the rat to
administered i.p. anesthesia of phenobarbital sodium (50
mg/kg body wt.) to expose the corpora cavernous.
-Then a 26 gauge needle connected to a polyethylene tube
(PE-50) with 100 U/mL of heparin on one side of the
Corpora cavernous is inserted for ICP measurement.
-Another 23 gauge needle is placed into the right carotid artery
connected to a PE-tube for the measurement of mean arterial
pressure (MAP).
-Both tubes should be connected to blood pressure transducers
which should also be connected via transducer amplifiers.

•Computers can be used to see real time display and recording of pressure
measurements (mm Hg).
•The cavernous nerve can also be stimulated by using a square pulse
stimulator connected to a platinum bipolar electrode positioned on the
cavernous nerve using 5 volts with a frequency of 50 Hz up to 5 min.
•The stimulation may be done three times and the ICP can then be
recorded. The ICP should be allowed to return to baseline before the next
stimulation.
•Statistically significant increase in ICP may imply their role on nitric
oxide (NO) and erectile function. The subsequent arteriolar dilation
leading to increased arterial inflow and impaired venous return (due to
engorgement of the cavernosum) builds up a pressure system within the
corpora that result in penile erection and rigidity.
54

Orientation behavior :
• Effects of aphrodisiac agent on behavior of male rats was gauged by evaluation of
three different parameters ,….
(1) Behavior towards female which involved licking and sniffing of female genital
organ,
(2) self exploratory behavior- which included non-genital grooming and genital
grooming, and
(3) general behavior which comprised of exploration, rearing and climbing. •
The behavior of rats was digitally recorded by using camera.
- The number of episodes of each of the behavior parameters would be counted.
Scoring is based on the episodes performed by the rats and every episode has certain
points

The scoring was given as ….
-0 (no sexual activity),
-1 (no interaction, rears and climbs on chamber),
-2 (sniffs other rat),
-3 (self- exploratory behavior i.e. , grooming and sniffing of
genitals),
-4 (grooms female rat anywhere),
-5 (rears and climbs sexually),
-6 (pursues and sniffs other rat),
-7 (tries to mount but easily discouraged),
-8 ( Mounts with an integrated deliberate manner, not easily
discouraged),
-9 ( reflex and almost involuntary mount)

Determination of hesitation time & attraction
towards female:
• A female rat was placed in a cage which had a wooden barrier of 15
cm separating male and female compartments which could be passed by
a motivated male rat.
• The hesitation time was recorded as the time (in sec) required by the
male rat before making an attempt to cross the barrier.
• In the same way, a scoring for attraction towards female was recorded
by a score between 0-5 during an observation period of 15 min.

Evaluation:
A complete cross of the partition by the male rat each time was
given a score of 5 while an attempt to climb was given a score of
2 and disinterest to climb was rated as 0.
-The readings were recorded on Days 0, 7, 14, 21, and 28 of
treatment.
• This test is useful in determining the willingness of a male rat to
cross an obstructive position, thus indicating the intent of sexual
attraction. Male rats of all the groups were subjected to
experimentation and their scores for attraction as well as hesitation
time were recorded.

Androgen Regulation of the Motor Copulatory Pattern in the
Male New Zealand White Rabbit
● The effect of castration and testosterone propionate (TP)
treatment on the motor copulatory pattern was studied in five
sexually experienced New Zealand white male rabbits.
● Five mature, sexually experienced rabbits were housed in
individual cages and supplied with food and water ad libitum.
● The rabbits were placed in an observation cage (110 cm
long, 60 cm deep, 50 cm high) and after an adaptation
period of 5 min presented with an estrous female. Sexual
partners were ovariectomized , sexually experienced New
Zealand white rabbits injected daily with estradiol
benzoate (10 ug).

● Prior to castration, the subjects were tested every third
day. The tests were terminated either at 30 min or after
five ejaculations.
● The animals were castrated under pentobarbital
anesthesia. Following castration, the rabbits were
tested every third day during the following
periods: 3-13, 30-40, 60-70, and 90-100 days.
● The animals were thereafter injected daily with
10 mg, SC, of testosterone propionate (TP) for
15 days, and tested for sexual behavior
beginning 3 days after the first TP injection.

Evaluation
● The following characteristics of the mounting pattern were
analyzed in all
● mounts:
-duration,
-frequency of pelvic thrusting, i.e., number of pelvic thrusts per
second;
-Intervals between the initiation of two successive pelvic thrusts
were measured in all mounts.
- The display of lordosis by females in response to mounting was
registered in the polygraphic record.

Improvement of sexual behavior and ejaculatory parameters by the
methanolic extract of Guibourtia tessmannii (Caesalpiniaceae) in high-fat-
diet-fed sexually sluggish male rats
● Guibourtia tessmannii (GT) (Caesalpiniaceae)
is claimed as a plant having aphrodisiac
property as per the African traditional medicine.
● Objective: This study evaluated the effects of
the methanolic extract of GT on sexual behavior
and fictive ejaculation in high-fat diet (HFD)-
induced sexually sluggish male rats.

● Guibourtia tessmannii (GT) is a
medicinal plant commonly used in
traditional medicine as sexual
enhancer.
-The aim of this study was to evaluate
the effects of the methanolic extract of
GT on sexual behavior and fictive
ejaculation in high-fat-diet-induced
sexually sluggish male rats
-GT improves sexual behavior by
reducing the ejaculatory latency and
increasing the sex drive.
-GT facilitates fictive ejaculation by
increasing the number of contraction
of the bulbospongiosus muscles after
urethral stimulation
-GT is a potential medicinal drug for the
management of sexual deficits
pertaining to obesity.

● Materials and Methods:
-Male Wistar rats fed either on HFD or standard diet (SD) for 16 weeks were
monitored for their growth rate and Lee index.
-At the end of this period, three consecutive copulatory tests were conducted
and HFD-induced sexually sluggish rats were selected.
-Besides Group 1 as time control (SD), drugs or vehicle (veh) were
administered orally everyday into three groups of rats as
-Group 2: HFD (veh),
-Group 3: HFD + GT (220 mg/kg), and
-Group 4: HFD + sildenafil citrate (5 mg/kg).
● Their copulatory activities were tested on day 1, 7, 14, and 21 and the
electromyography of bulbospongiosus muscles was assessed for fictive
ejaculation on day 22.
conclusion: These findings provide robust evidence for the GT treatment in the
management of sexual deficits pertaining to obesity.

•Biochemical studies
A) Determination of testicular and serum cholesterol
-Cholesterol is the precursor in the synthesis of many physiologically important
steroids such as bile acids, steroid hormones and vitamin D and its requirement
for normal testicular activity has been well established.
• Testicular and serum cholesterol concentrations may be determined by the
Chod-PAP method as Briefly, 0.02 ml of the sample is mixed with 2.00 ml of
the working reagent and the absorbance of the resulting mixture is taken after 5
min. at 546 nm.
• A potential for aphrodisiac should result in statistically significant increase in
testicular and serum cholesterol concentration.
• if increase, may cause stimulation in the steroid genesis, which may lead to
increased testosterone concentration. If increase in testosterone concentration
should increase in libido.
66

B) Hormonal determination assay
• The positive effects of male sexual behavior must have been brought
about by to identify level of some reproductive hormones like
testosterone, luteinizing hormone (LH), follicle stimulating hormone
(FSH) and prolactin.
• Testosterone supplementation has been shown to improve sexual
function and libido.
• Luteinizing hormones (LH) and Follicle Stimulating Hormone (FSH)
produced by anterior pituitary lobe, they are necessary for maintaining
testosterone levels. An increase in the concentrations of LH and FSH
should normally increase the testosterone concentration.
• Normally, prolactin is made by specialized pituitary cells called
lactotrophs. Prolactin increases the production of breast milk and
suppresses secretion of LH and FSH. The role of prolactin in men is not
known.
67

• if high levels of prolactin in men, may cause hypogonadism, low blood
testosterone levels and decrease in sex drive (libido) and sexual function.
• aphrodisiac agents shows reduction in the concentrations of
prolactin in males which would enhance the levels of LH and FSH
and by extension the testosterone concentration.

C) Fructose content in seminal vesicles
•The seminal vesicles were macerated with 3 mL of distilled water
and centrifuged at 4000 r.p.m. for 12 min.
•To the supernatant, fluid collected after centrifugation add 0.5 mL
of resoreinol and 1.5 mL of HCl.
•The mixture was kept at 80 for 12 min. The reaction with resorcinol
developed a rosy colour, which was measured at 500 nm using
spectrophotometer.
•A calibration curve was drawn using dilutions of fructose solution
and measument of the colour developed with resorcinol and HCL.
69

E. )In-vivo sperm count :
• Epididymis of rats of each group were homogenized and taken into 5 mL of
1% sodium citrate solution and squashed thoroughly with the help of needle
and forceps until a milky suspension was obtained.
• The solution was filtered through 80 m mesh and the volume was made up to
10 mL with the same solution; the made up volume was inclusive of washings
of the filter.
• The suspension was shaken thoroughly and the spermatozoa were counted in
five WBC counting chambers of the haemocytometer. The average numbers
of sperms per chamber are reported.
Evaluation
-The progressive sperm motility,
-sperm count,
-live/dead ratio (viability) and
-morphology were determined.

ASSESSMENT OF SPERM
VIABILITY AND MORPHOLOGY
• A viability study (percentage of live spermatozoa) was done using
eosin/ligroin stain. A drop of semen was squeezed onto a
microscope slide and two drops of the stain were added.
• Thin smears were then prepared and observed under a light
microscope at 100 magnification. Viable sperm remained colorless
while non-viable sperm stained red.
• The stained and the unstained sperm cells were counted using
40/100 microscope objectives and an average value for each was
recorded from which percentage viability was calculated.
• To determine the percentage of morphologically abnormal
spermatozoa, the slides stained with eosin–ligroin (5 slides/rat)
viewed under a light microscope at 100 magnifications. 71

Refrences
•Article journal homepage: www.jadweb.org
•http://www.wikipedia.com
•https://reader.elsevier.com/reader/sd/pii/S2221618913601055?token=9686312D6857347
38BEECB1083A9AFC92B685AFE166C7933301D0A3F189C01209607BB7D2D8AA9E4F3D811
E47970710C
.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2269215/
.https://www.jove.com/video/3954/rat-mesentery-exteriorization-model-for-
investigating-cellular
•https://www.researchgate.net/publication/257746599_An_overview_of_the_current_met
hodologies_used_for_evaluation_of_aphrodisiac_agents
•https://sci-hub.tw/10.1016/0018-506x(80)90027-6
•http://www.phcog.com/article.asp?issn=0973-
1296;year=2019;volume=15;issue=62;spage=54;epage=61;aulast=Defo
•http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842002000400008
72

Screening of ANTIFERTILITY AGENTS and APHRODISIACS

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