Based on the cytology provided, the bitch was likely bred during estrus and therefore has a high chance of pregnancy since she was bred at a very fertile time in her cycle. The smears from Tuesday-Thursday show 100% cornified cells indicating estrus, and she was bred only 2 days prior to the first smear, well within the fertile window.

1. CANINE VAGINAL
CYTOLOGY

2. Canine Vaginal Cytology
What causes the change in cytology?
How do you take a sample?
How do the cells look like?

3. Demystifying Vaginal
Cytology

4. Vaginal Cytology
1. Moisten a long cotton-
tipped wooden applicator
with a few drops of sterile
saline.
2. Lift the dorsal aspect of the
vulva and insert the swab at the
dorsal aspect. Point posterior
somewhat and make a ‘dimple’
between the rectum and dorsal
vulva.

5. Vaginal Cytology
3. Push and rotate the swab in dorsally. It
takes more pressure than many realize.
If it does not go in easily, back out a little
and spin it, as it may be caught on a
vaginal fold. Do not go too far ventral or
you will go into the bladder.
Good!

6. Vaginal Cytology
Bad!

7. Vaginal Cytology
Cells taken from the
vestibule or caudal vagina
may not represent the
true cellular population.

8. Slide Prep and Stain
Roll the swab out onto a slide.
Roll in different ‘streaks’ down
the slide.

9. At the end of anestrus and during proestrus, the estrogen
from the follicles is rising and peaks just before the
beginning of estrus.

10. It is cytology to change. If there is no estrogen,
the vaginal cytology does not he ESTROGEN that
causes the vaginal change.

11. Without ESTROGEN, (during anestrus and diestrus)
the vaginal cytology is non-cornified

12. Without ESTROGEN, the
vaginal epithelium is very
thin and the cytology is
non-cornified.
With ESTROGEN, the
vaginal epithelium is
very thick and
hyperplastic and the
cytology is cornified.
Without ESTROGEN,
(even if progesterone
is high as in diestrus)
the vaginal epithelium
is very thin and the
cytology is non-
cornified.

13. Cell types
Cornified
vs
Non-cornified

14. Parabasal
Intermediate
Superficial
Anuclear
Parabasal cells
have round
cytoplasm and a
stippled nucleus
Intermediate cells
have more angular
cytoplasm and a
stippled nucleus
Superficial cells
have an angular
cytoplasm and a
non-stippled or
pyknotic nucleus
Anuclear cells
have an angular
cytoplasm and
no visible
nucleus

15. Parabasal
Intermediate
Superficial
Anuclear
Cornified
Non-cornified
Parabasal cells
have round
cytoplasm and a
stippled nucleus
Intermediate cells
have more angular
cytoplasm and a
stippled nucleus
Superficial cells
have an angular
cytoplasm and a
non-stippled or
pyknotic nucleus
Anuclear cells
have an angular
cytoplasm and a
no visible nucleus

16. Parabasal
Intermediate
Superficial
Anuclear
Cornified
Non-cornified

17. Parabasal
Intermediate
Superficial
Anuclear
Cornified
Non-cornified
This should also
help you
remember what
the cells look like.
Remember, if the
nucleus is there,
but not stippled,
the cell is cornified.
If the nucleus is
stippled, the cell
is non-cornified.

18. P
I
S
A
S
A
A
A
I
*
*
*
*
*
*
*
*
*
*
*
*
I
S
A
*
*
*
Can you find the P’s?
Identify and click.
Can you find the I’s?
Identify and click.
Can you find the S’s?
Identify and click.
Can you find the A’s?
Identify and click.
Can you find the RBC’s?
Identify and click.
The resolution of photos makes it difficult to identify the stippling. It is easier
looking through the microscope. There is some judgment involved too.
Cornified and non-cornified are the big differences to note.

19. During anestrus and diestrus, the
cornification is minimal

20. Vaginal cytology is an
estrogen assay. If estrogen is
low, the epithelium is thin
and very few cells, non-
cornified cells can be
obtained

21. Shows the swab being pushed back and forth, in
reality it is rolled. The top is a cartoon and the
bottom is a photomicrograph of a non-estrual
bitch.
Note the epithelium is thin and not many cells
are obtained when they female is non-estrual
(anestrus or diestrus).

22. Shows the swab being pushed back
and forth, in reality it is rolled onto
the slide.

23. Notice how few cells are on the slide

24. Here is glass slide
from a non-estrual
bitch. Notice that
there are not many
cells.
Here is
photomicrograph
from a non-estrual
bitch. Notice that
there are not many
cells, they are non-
cornified (see the
stippling?), and
there is lot of
‘junk’ in the
background.

25. During proestrus the cornification rises about 10%/day, until
estrus when it reaches 100%. It is 100% cornified throughout
estrus. The cornification abruptly drops to <50% on the first day
of diestrus.

26. Vaginal cytology is an estrogen assay. If estrogen
is high, the epithelium is thick and many cornified
cells can be obtained

27. Shows the swab being pushed
back and forth, in reality it is
rolled. The top is a cartoon and
the bottom is a
photomicrograph of a non-
estrual bitch.
Note the epithelium is thick
and hyperplastic. Many cells
are obtained when the female
is estrual (proestrus or estrus).

28. Notice how many cells will be on the slide.

29. Here is a glass slide
from an estrual
bitch. Notice that
there are many cells.
Here is a
photomicrograph
from an estrual
bitch. Notice that
there are many cells,
they are cornified,
and the background
is clear.

30. What kind of cell is this?
Parabasal Intermediate Superficial Anuclear
Incorrect
Do you see a
nucleus?
What is the
shape of the
cytoplasm?
Incorrect
Do you see a
pyknotic
nucleus?
Correct
Notice the
lack of
nucleus and
the angular
cytoplasm.
Incorrect
Do you see a
nucleus?
What is the
shape of the
cytoplasm?

31. What kind of cell is this?
Parabasal Intermediate Superficial Anuclear
Incorrect.
Do you see
the pyknotic
nucleus?
The
cytoplasm is
angular.
Correct.
You see a
pyknotic
nucleus and
angular
cytoplasm.
Incorrect.
Notice the
pyknotic
nucleus.The
cytoplasm is
angular
however.
Incorrect.
Do you see
the pyknotic
nucleus?
What is the
shape of the
cytoplasm?

32. What kind of cell is this?
Parabasal Intermediate Superficial Anuclear
Correct.
Notice the
stippled
nucleus
and round
cytoplasm.
Incorrect.
Do you see
the stippled
nucleus? It
is not
pyknotic.
The
cytoplasm is
rounded
and not
angular.
Incorrect.
Do you see
the stippled
nucleus? It
is present.
The
cytoplasm is
rounded
and not
angular.
Incorrect.
The nucleus
is stippled,
but the
cytoplasm is
not angular.

33. What kinds of cells are these?
Parabasal Intermediate Superficial Anuclear
Incorrect.
Notice the
stippled
nucleus but
the
cytoplasm is
more
angular. A
judgment
call.
Incorrect.
Do you see
the stippled
nucleus? It
is not
pyknotic.
The
cytoplasm is
somewhat
angular.
Incorrect.
Do you see
the stippled
nucleus?
The
cytoplasm is
Somewhat
angular.
Correct
The nucleus
is stippled,
and the
cytoplasm is
angular.

34. Are these cells cornified or non-
cornified?
Cornified Noncornified
Incorrect.
Notice the
stippled
nucleus.
Cornified
cells do not
have a
stippled
nucleus
Correct.
The nucleus
is stippled.

35. Are these cells cornified or non-
cornified?
Cornified Noncornified
Incorrect.
Notice the
stippled
nucleus.
Cornified
cells do not
have a
stippled
nucleus
Correct.
The nucleus
is stippled.

36. If you saw this slide, what stage of
estrus would you place her?
Anestrus Proestrus Estrus Diestrus
Incorrect.
Notice all
the cells are
cornified.
The nuclei
are
pyknotic.
Correct.
Notice all
the cells are
cornified.
The nuclei
are
pyknotic.
Incorrect. Notice
all the cells are
cornified. The
nuclei are pyknotic.
In diestrus there is
a mixture of
cornified and non-
cornified cells.
Incorrect.
Notice all the
cells are
cornified. The
nuclei are
pyknotic. In
proestrus there
is a mixture of
cornified and
non-cornified
cells.

37. If you saw this slide, what stage of
estrus would you place her?
Anestrus
Proestrus
Estrus
Diestrus
Incorrect.
Notice some
of the cells are
cornified.
There are
many cells
present.
Incorrect.
Notice some of
the cells are
non-cornified.
Maybe. Notice
about half the
cells are
cornified. It
could be
diestrus, but
what else could it
be?
Maybe. Notice
about half the cells
are cornified. It could
be proestrus, but
what else could it be?

39. ?

41. THANK YOU

42. Case 1
 Classify stages each as being proestrus,
estrus or diestrus of a cycle by examining
images from 8 smears taken from a 3 year-
old bitch which has a swollen vulva and to
be spotting blood. A series of vaginal smears
were taken, the first labeled as Day 1.

43. Day 1: Proestrus- Intermediates,
superficials and red blood cell

44. Day 2: Estrus- superficials and
red blood cells

45. Day 3: Estrus- superficials

46. Day 6: Estrus- all superficials

47. Day 9: Estrus- all superficials

48. Day 10: Estrus- superficials and
red blood cells

49. Day 11: Diestrus- Abrupt change to
intermediates and parabasals
(WBC,RBC)

50. Day 12: Diestrus Day 2-(Confirmation
of day 1)

51. CASE 2
 A bitch was observed with vulvar swelling on
Friday and strongly attracted the neighbor's male.
The owner has prepared a vaginal smear Friday
night and again on Sunday. On Monday, they took
the bitch to the stud and she was bred without
difficulty.
 After the breeding, the owner ask you to examine
the smears prepared on Friday and Sunday. Based
on those observations, would you say that the
bitch is likely to deliver pups? Why or why not?

52. Friday's Slide

53. Sunday's Slide

54.  Ans: The bitch is likely to be pregnant. She was clearly
in cytologic estrus on Friday(100%superficial cells)but
in diestrus on Sunday (only one superficial cell with
remainder mostly small intermediates). Breeding took
place on Monday which was either diestrus day 2 or 3
and past the normal period of fertility.

55. CASE 3
 On Sunday there had been a tie between a bitch and an intact
male. But the bitch had not shown any signs of coming into
heat.
 As the owner did not want her to be pregnant, so on Tuesday
he started preparing vaginal smears to see whether the bitch
was in her cycle.
 So here's the question: What are the chances that the bitch
became pregnant? Assume that the male was fertile. Base your
answer on when she was bred relative to progression of vaginal
cytologic changes. Remember that the breeding took place on
Sunday, two days before the first vaginal smear.

56.  Tuesday: Estrus, essentially all are cornified superficial
cells

57.  Wednesday: Estrus, essentially 100% cornified

58.  Thursday: Estrus, essentially 100% cornified

59.  Friday: Diestrus day 1, note the dramatic change to
nucleated cells

60.  Saturday (last slide): Diestrus day 2

61.  Ans: The bitch was in a breeding tie 5 days prior to
diestrus which is considered a very fertile time. Also,
the smear taken on Tuesday showed full cornification
also suggesting that she was bred during estrus.
Palpation roughly 20 days after the onset of diestrus
revealed that, it was pregnant.