Experimental evaluation of antifertility agents The document discusses various in vivo and in vitro methods to evaluate potential antifertility agents in males and females. It describes tests to determine if compounds interfere with ovulation, implantation, fertilization or early embryo transport in females. For males, it outlines assays to test if agents affect spermatogenesis, sperm function or fertility. The document also provides protocols for characterizing the estrogenic, progestational, androgenic or anti-hormonal properties of test compounds. It details rat, rabbit and chicken models commonly used to screen for contraceptive activity and hormonal effects.

1. Experimental evaluation of
antifertility agents
June 25, 2020

2. • Definition: Agents that prevent fertility by
interfering with various normal reproductive
mechanisms, in both males and females

3. Characteristics of an ideal
contraception
• 100% effective
• Safe
• Easy to use
• Effects should be reversible
• Aesthetically, personally, socially and
politically acceptable
• Readily available and legal

4. Methods for females
Inhibition of ovulation
Interference with implantation
or dislodging the implanted
early embryo
Interference with transport of
ova
Prevention of fertilization

5. Anti-ovulatory activity
Anti-ovulatory
activity
hCG induced
ovulation in rats
Cupric acetate
induced ovulation in
rabbits.

6. 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
Principle: Injection of anti ovulatory drugs, prior
to induction procedure will prevent ovulation

7. Procedure
Immature female albino rats 24-26
days of age are used and treated with
the test drug
After the administration of the test
drug, hCG is given exogenously for
ovulation.
After 2 days ,animals are sacrificed
,ovaries are dissected out, and subjected
to histopathological evaluation .
The results are compared with the
control group

8. Cupric acetate-induced ovulation in
rabbits :
• Rationale : 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 1 % cupric
acetate in 0.9 saline).
• Injection of anti ovulatory drugs, 24 hours before
the induction procedure prevents ovulation.

9. Procedure
Sexually mature female rabbits, 3-4 kg
are kept in isolation for at least 21 days
Treated with test drug and 24 hours
later cupric acetate. 24 hours later -
sacrificed and ovaries are examined
The total number of ovulation points
on both ovaries are recorded for each
animal. The ovaries and uterus are
examined histopathologically.

10. Macroscopic features of rabbit ovaries at day 7 after the i.m. injection of (A)
20 mg of gonadoreline (GnRH); (B) 24 mg murine b-nerve growth factor
(NGF) or (C) 1 mL of saline solution (SS) with (ICþ) or without (ICÀ)
catheter introduction into the vagina. In the picture, black arrows indicate
corpora lutea and white asterisks show haemorrhagic follicles.

11. Estrogenic activity

12. Estrogenic activity
In-Vivomethods
Vaginal opening
Assays for water uptake
Four day uterine weight assay
Vaginal cornification
Chick oviduct method

13. Vaginal opening method
Principle: vaginal opening occurs in immature
female albino mice & rats by treating with
estrogenic compounds. The sign of complete
vaginal opening is observed as a sign of
estrogenic activity.

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

15. Assay for water uptake
Principle: uterus responds to estrogen by uptake
and retention of water.

16. Procedure
Immature 18-22 day female rats
selected, divided into control and test
groups
Control group (cotton seed oil ) and
test group (test drug) in a graded
manner
5 hours later animals are sacrificed,
uteri removed & weighed
Desiccated at 100C and reweighed.
Difference in the % of weight gain is
calculated

17. Four day uterine weight assay
• Principle: estrogen causes increase protein
synthesis and thus weight gain. Peak affect is
observed in 40 hours.

18. Procedure
Immature 18-22 day female rats
selected, divided into control and test
groups
Control group (cotton seed oil ) and
test group (test compound ) in a
graded manner
Day 4 animals are sacrificed, uterine
contents are removed & weighed
Desiccated at 100C and reweighed.
Difference in the % of weight gain is
calculated

19. Vaginal cornification
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
Principle: If a drug has estrogenic activity it will
cause the animal to skip other stages and go into
estrus stage

20. Stages of estrus cycle in rats
Proestrus
Nucleated epithelial cells
Estrus
Squamous cornified
cells (ovulation)
Metestrus
cornified+leukocytes
Diestrus
leukocytes

21. Rat vaginal smear-estrus

22. Chick oviduct method
• Weight of the oviduct of young chicken is
increased dose-dependent by natural and
synthetic estrogen

23. Pullet chicken divided in control and
test groups (6-10 each)
Control (0.02-0.5 μgm estradiol) & test
drug (increasing doses of test drug)-
injected twice daily till day 6
Day 6 animals are sacrificed, body and
oviduct weighed

24. In-vitro
• Estrogenic receptor binding assay
• Dextran coated charcoal (DCC) adsorption
technique

25. Anti estrogenic activity
In vivo
Antagonism
of
physiological
effects of
estrogen
In Vitro
Aromatase
inhibition

26. Anti estrogenic activity
• Antagonism of physiological effects of
estrogen-
– Water uptake of uterus
– Uterotrophy
– Vaginal cornification

27. Aromatase inhibition
• Compounds that inhibit aromatase have anti
estrogenic activity
DHEA
Androestenedione
Estrone
Aromatase
Testosterone

28. Progestational activity
In-Vivomethods
Pregnancy maintenance
Proliferation of uterine endometrium in
estrogen primed rabbits
Carbonic anhydrase activity in rabbit’s
endometrium
Deciduoma reaction in rats
Prevention of abortion in oxytocin treated
pregnant rabbits

29. In vitro methods
• Progesterone receptor binding assay

30. Pregnancy maintenance
• Principle: Progesterone maintains pregnancy
• Procedure-
All pregnant
animals
Control
Average living
fetus
Test group
(ovariectomy)
Average living
fetus

31. Proliferation of uterine endometrium in
estrogen primed rabbits
Female rabbits
weighing 800-
1000gms
Primed with estradiol
f/b progestational
compound
Endometrium-
secretary phase

32. Procedure
Female rabbits 800-1000gms, inj.
Estradiol 0.5 μgm/ml daily
Day 7- drug treatment is started, 5
doses for 5 days
24 hours after the last dose – animals
sacrificed- mid section of uteri frozen
and histologaically examined
Glandular development is graded 1-4

34. Carbonic anhydrase activity
• Principle: linear relationship between
progestogens and carbonic anhydrase activity
Immature female albino rabbits
Control
After sacrifice, endometrial
extract of the uterus is evaluated
for carbonic anhydrase activity
Test
estradio
l

35. Deciduoma reaction in rats
Principle: maternal/placental tumor formation
by progestational drugs in traumatized uterus of
ovariectomized rats

36. Ovariectomized adult female albino
rats 150-200 gms, primed with
estradiol
Day 1 to 9- drug treatment is given.
On day 5, one uterine horn is injured
using histamine
24 hours after the last dose – animals
sacrificed- mid section of uteri frozen
and histologaically examined
Glandular development is graded 1-4

37. Prevention of abortion in oxytocin
treated pregnant rabbits
• Principle: i/v oxytocin in pregnant rabbits on
30th day causes abortion
• Prior administration of progestational
compound prevents this response

38. Control group
Pregnant rabbits
Day -30 Oxytocin 10 U
(24 hours after oil)
No. of abortions within
2-30 minutes
Test group
Pregnant rabbits
Day -30 Oxytocin 10 U
(24 hours after test drug)
No. of abortions within
2-30 minutes

39. Anti progestational activity
• Anti progestational activity in immature
rabbits (Mc Ginty Test)
• Principal :
• 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 .

41. Procedure
Positive control Test group Negative control
All the rabbit are primed with estradiol -6 days. Upper section
of each horn is ligated without compromising the blood supply
Right
horn
Left horn Right
horn
Left horn Right
horn
Left horn
Progester
one
Vehicle
0.5%CM
C
Progester
one
Progester
one+ test
drug
Progester
one
Vehicle
Animals with estradiol will have increased weight

42. Anti implantation activity
• The anti-implantation activity is expressed as the
percentage of animals showing absence of
implantations in uteri.

43. Day 10- laprotomy to count number of implants in
each horn and number of corpora lutea in each ovary
This is designated as day 1 of pregnancy and those rats
were divided into five groups containing six rats in
each group.
Rats found in proestrus phase of cycle were caged
with males of proven fertility, 3:1 and examined for
evidence of copulation.
Vaginal smears from each rat were monitored daily
and the rats with normal estrous cycle were selected.

44. Anti implantation activity

45. Figure 8. - Effect of antisense oligodeoxynucleotide on embryo implantation. (A) Two
representative uteri of D7 (n=60). (D7a) Control group (CON) (n=30), pregnant mouse
was injected in both horns with DD water. (D7b) Treatment group (n=30), pregnant
mouse was injected with IK cytokine A-ODNs in the left horn, with IK cytokine S-
ODNs in the right horn on Day 3 of pregnancy. (B) Statistical analysis of implanted
embryo numbers in the uteri with different treatments (*P<0.05).

46. %postimplant losses =
No of implants−No of litters
No of implants
× 100
%Antifertility rate =
No of CL−No of litters
No of CL
× 100

47. Abortifacient activity
Adult female rabbits
Date of mating is day -1 of pregnancy
Day 12 – pregnancy is confirmed by palpation
Day 20-intra amniotic/ intraplacental administration of
abortifacient drug
Effect- vaginal bleeding, changes in weight, abdominal
palpation, post mortem examination

48. Methods for males
• Interference with spermatogenesis without loss
of libido or secondary sexual characters

49. Methods for males
In vivo
Cohabitation test
Fertility test
Subsidiary test
In Vitro
Spermicidal activity
Immobilization assay
Non specific
aggregation estimation
Sperm revival test

50. Cohabitation test-
Principle: time interval for litter production after
placing treated males with 2 females. Date of
mating is calculated from date of parturition.
Subsidiary test-
Principle: change in sperm count over time.

51. Fertility test
• Principle: Evaluation of average litter size
5-10 male rats with proven fertility,
treated with test drug, paired with fertile
female, 1:3
Daily vaginal smears examined for
sperms. All females that pass through
estrus cycle are assumed to have mated
Mated females are kept separately till
litter is delivered

52. Evaluation:
• Average litter size =
total no. of litters
No of females mated
• If vaginal smear shows leukocytes for 10-14
days- pseudopregnancy is confirmed.
– Loss of libido
– Aspermic copulation

53. Androgenic activity
• Chicken comb method
• Weight of ventral prostrate, seminal vesicles
and musculus levator ani
• Nitrogen retention

54. Chicken comb method
• Chicken comb growth depends on androgenic
compounds
Control group (n=8) Test group (n=8)
Individual capons (castrated cocks), comb measured.
Daily placebo (olive oil)
i.m. 5 days
Daily test compound i.m. 5
days
Comb is measured 24 hours after the last dose

55. Weight of ventral prostrate, seminal
vesicles and musculus levator ani
• Principle: androgen affect all secondary sex organs
Control group Test group
Immature male rats 55gms, orchiectomized
Placebo/0.5%CMC/0.2ml
sesame oil 10 days
Test compound 10 days
Only placebo or vehicle Testosterone s/c 0.02,
0.1, 0.5mg/ animal
Day 11- animal sacrificed and ventral prostrate,
seminal vesicles and musculus levator ani weighed

57. Nitrogen retention
• Principle: anabolic agents induce positive
nitrogen balance in rats

58. 24 hours urine sample is collected 3 times a week for
nitrogen
Placebo Test drug
300 gms
Liquid diet rich in nitrogen is forced fed for 30 days
25 day rats selected. Castrated
67 days normal diet

59. Anti androgenic activity
• Chicken comb method
• Antagonisim of effect of testosterone on
weight of ventral prostate, seminal vesicles
and musculus levator ani
• Anti-androgenic activity in female rats

60. Chicken comb method
• Chicken comb growth depends on androgenic
compounds
• Female chicken are used and change in comb
weight is measured

61. Animals sacrificed 24 hours later, combs removed and
weighed
For next 4 days
Placebo(0.1ml s/c) Test drug (0.1ml s/c)
Testosterone given in finely ground chick mash (80mg/kg food)
1-3 day female leghorn chicken used

62. Antagonism of effect of testosterone
on weight of ventral prostate, seminal
vesicles and musculus levator ani
• Principle: growth of ventral prostate, seminal
vesicles and musculus levator ani is stimulated
by testosterone and inhibited by anti-
androgenic compounds

63. Animals sacrificed day 8, ventral prostate, seminal
vesicles and musculus levator ani removed and
weighed
For next 7 days
Testosterone
only(0.1ml s/c)
Testosterone with test
drug
Male rats, 50-70 gms, castrated

64. Anti-androgenic activity in female rats
Principle: antagonism of the anti-androgen
against the tropical effect of testosterone on
uterine and preputial growth

65. Animals sacrificed day 13, uteri and preputial glands
removed and weighed
For next 12 days
Testosterone
only(0.1ml s/c)
Testosterone with
antagonist
Female rats, 40-45 gms, ovariectomized

66. References
• SK Gupta. Antifertility agents. Drug screening and
methods. In: Jaypee publishers. Ed 3: 132-51
• β-nerve growth factor identification in male rabbit
genital tract and seminal plasma and its role in
ovulation induction in rabbit does b-nerve growth
factor identification in male rabbit genital tract and
seminal plasma and its role in ovulation induction in
rabbit does. Italian Journal of Animal Science · Oct
2017DOI: 10.1080/1828051X.2017.1382315
• Kafkas Univ Vet Fak Derg 2017; 23: 137-144, DOI:
10.9775/kvfd.2016.16008

67. Thank you