1. PROFESSIONALISM “No man ever reached to excellence in any one art or profession without having passed through the slow and painful process of study and preparation.” -Horace

2. THE ESTROUS CYCLE

3. NORMAL ESTOUS CYCLE: THE BITCH Estrous Cycle: Defined as the time from the beginning of one heat period to the beginning of the next. Onset of puberty: 6-24mths; Avg.: 9- 10mths Follicular phase (proestrus and estrus) or the Luteal phase (diestrus and anestrus).

4. ESTROUS CYCLE – Proestrus : leading to estrus (Avg: 9-10 days) – Estrus: MATING (Avg: 9-10 days) – Metestrus: 2 phases in diestrus – Diestrus: Pregnancy is established (Avg: 57-58 days) – Anestrus: time when no reproductive event (Avg: 2-5 months) PAGE 374 (Figure 14-1)

5. HORMONE Hypothalamus GnRH Anterior Pituitary FSH Ovarian Follicle Estrogen Causes GnRH release > LH surge

6. HORMONE CHANGES Estradiol concentration increases as ovarian follicles mature and the uterus enlarges

7. METETRUS

8. Cycle StageLengthHormonal changesPredominant Cell TypesErythrocytesNeutrophil s Behavior of the Bitch Clinical Signs Proestrus~9 daysEstrogenEarly: mixed population of cells Late: large intermediate and superficial cells Early: present Late: ± Early: present Late: decreased Attractive to the male, but won’t stand for mating Vulvar edema and swelling, bloody discharge Estrus~9 daysLH (surge) Estrogen Progesterone 90% superficial cells Bacteria usually present ±AbsentAccepts male and will stand Less edema, discharge becomes clearer Diestrus~60 daysProgesteroneAbrupt change in relative # of epithelial cells. Superficial cells decrease by 20% ±Few to none Ceases to accept male Little discharge, edema decreased AnestrusDepends on whether pregnant or not. 70-80 days for CL to regress and 130 days to repair endometrium. ProgesteroneParabasal and Intermediate cells Few if anyFew if present No outward signs Scanty, tenacious secretions

9. Typical morphological features of the vaginal mucosa and endometrium in anestrus (A), proestrus (P), estrus (E), and early diestrus (D). H&E. Original magnification 200X.

11. NORMAL ESTROUS CYCLE: PROESTRUS PROESTRUS: the period when the bitch is sexually attractive, but rejects the male’s advances Physical signs of proestrus: – Serosanguinous vaginal discharge – Vulvar swelling – Vaginal edema – Internal physical signs: Lengthening and hyperemia of the uterine horns Enlargement of the cervix Thickening of the vaginal wall

12. PROESTRUS http://www.vetmed.wisc.edu/data/coursematerial/bosu/CR-lecture1and2.pdf vulvar edema, hyperemia, and a serosanguineous discharge

13. PROESTRUS Hormonal signs of proestrus: – Increasing serum concentrations of estradiol (estrogen) estrogen is responsible for the physical signs of proestrus Estrogen is being produced by the maturing follicles in the ovary (under the influence of Follicle Stimulating Hormone or FSH) As proestrus progresses, the estrogen levels begin to peak and initiate the onset of estrus.

14. PROESTRUS Cytologic signs of proestrus: – Vaginal Cytology Parabasal cells and intermediate cells predominate; wbc’s and rbc’s present Duration of proestrus: 3- 17 days; Avg.: 9 days

15. VAGINAL CYTOLOGY A saline moistened cotton swab is inserted through the vulvar lips into the vagina. The swab is angled craniodorsally to avoid the clitoral fossa. A clean otoscope may be used to guide the swab and to provide a light source for visual guidance. Once the swab is located cranial to the urethral orifice, the swab is rotated slightly to exfoliate and collect the cells.

16. VAGINAL CYTOLOGY The cells are transferred to a clean glass slide by rolling the swab along the surface of the slide. Intact cells are obtained during transfer by rolling the swab. In contrast, back and forth smearing of the swab to transfer the cells will cause sever cellular distortion and rupture. Once the cells have been transferred, the slides are air dried, stained with Romanowsky or new methylene blue stains, and examined microscopically

17. VAGINAL CYTOLOGY Romanowsky stains (Wright, Giemsa, Leishman, Diff-Quik®), new methylene blue, toluidine blue, trichome stains (Papanicolaou, Shorr, Sano) and hematoxylin and eosin (H&E). 1 Personal experience, preference, cost, and ease of use usually determine the choice of stain. In any event, the stain should be economical, easily stored, and produce consistent staining that is adequate for cytologic needs

18. Proestrus Early proestrus with a mixture of nondegenerate neutrophils; parabasal, intermediate, and superficial epithelial cells, erythrocytes, and mucus (Wright- Leishman stain). Late proestrus with erythrocytes, scattered superficial cells, and absence of neutrophils (Wright- Leishman stain). erythrocytes account for most of the cells observed on vaginal smears

19. THE NORMAL ESTROUS CYCLE: ESTRUS ESTRUS: The period of acceptance of the male Physical signs of estrus: – The swollen vulva is less turgid – The vulvar discharge is usually less bloody – The bitch allows mating “standing” heat “flagging” behavior (tail deviated to the side)

20. ESTRUS Hormonal signs of estrus: – Serum estrogen peaks in late proestrus and early estrus. The estrogen peak initiates a surge of luteinizing hormone (LH) LH results in ovulation (rupture) of the mature follicle and release of the oocyte. LH also stimulates the cells left behind in the empty follicle to multiply into the corpus luteum (See metestrus) This stage of the reproductive cycle is influenced mainly by estrogens – serum progesterone levels begin to increase toward the end of estrus (and beginning of metestrus) Progesterone is produced by the corpus luteum which is forming from the empty follicle (see metestrus) Progesterone is the hormone that maintains pregnancy

21. ESTRUS Cytologic signs of estrus: – Vaginal cytology: Superficial and superficial anuclear squamous cells predominate (aka cornified cells); rbc’s usally present, wbc’s usually absent Duration of estrus: 3-21 days; Avg.: 9 days This stage of the reproductive cycle is difficult to determine cytologically because distinguish features are only present microscopically at the beginning and the end of estrus

22. ESTRUS: VAGINAL CYTOLOGY CORNIFIED EPITHELIAL CELLS Estrus characterized by superficial cells that are keratinized, largely anucleate, and have angular, folded cell margins (Wright-Leishman stain).

23. THE NORMAL ESTROUS CYCLE: METESTRUS METESTRUS: The period after ovulation when the corpus luteum develops. – Internal signs of metestrus: Lining of the uterus is thick and “juicy” in preparation for implantation of a fertilized ovum Loss of the cornified epithelial lining that developed in the vagina during proestrus and estrus

24. THE NORMAL ESTROUS CYCLE: DIESTRUS DIESTRUS: the period when the corpus luteum has reached maximum size and effectiveness. Begins with the bitch’s refusal to mate – Physical signs of diestrus: None except the cessation of signs of estrus – Hormonal signs of diestrus: Serum progesterone levels continue to increase and peak during diestrus In a pregnant bitches, there is a rapid pre-partum drop in progesterone ~24 hours before partuition In non-pregnant animals, the decline may be more gradual

25. DIESTRUS Cytologic signs of diestrus: – Vaginal cytology: A sudden decrease in the number of superficial cells and a reappearance of intermediate and parabasal cells, and wbc’s. Duration of diestrus: ~2mths – Dogs and cats are unique in that their corpora lutea appear to have preprogrammed life spans.

26. DIESTRUS: VAGINAL CYTOLOGY INTERMEDIATE CELLS PARABASAL CELLS

27. DIESTRUS PSEUDOPREGNANCY: – A prolonged diestrus – Animal may show signs of pregnancy: Nesting behavior Mammary enlargement – Lactation in extreme cases Clinical signs fade with time

28. THE NORMAL ESTROUS CYCLE: ANESTRUS ANESTRUS: Follows diestrus and ends with the onset of proestrus; criteria vary – First time plasma progesterone reaches a level below 3 nmol/L – The moment that the influence of progesterone on the endometrium is no longer evident. Cytologic signs of anestrus: – Samples are almost acellular – The cells present are parabasal cells and small intermediate cells. Duration of anestrus: Avg.: ~4.5mths.

29. THE NORMAL ESTROUS CYCLE: THE QUEEN Onset of puberty: 6-9mths Female cats are seasonally polyestrous – Cycle controlled by the photoperiod and body weight 12-14 hours of light and 2.5kgs – In the absence of ovulation or pregnancy, repeated estrous cycles occur every 10-14 days

30. THE NORMAL ESTROUS CYCLE: PROESTRUS in the queen PROESTRUS: The period of follicular development, but refusal to mate – Physical signs of proestrus: Owner may notice increased affection No vulvar swelling or vaginal discharge in cats – Hormonal signs of proestrus: Characterized by increasing serum estrogen concentrations – Cytologic signs of proestrus: Epithelial cells become cornified – Duration of proestrus: 1-2 days

31. THE NORMAL ESTROUS CYCLE: ESTRUS in the queen ESTRUS: The period of sexual receptivity and follicle maturation – Physical signs of estrus: Queen allows copulation Extreme affection (head rubbing, vocalization, crouching of the thoracic limbs with rigid extension of the pelvic limbs, lordosis, lateral deviation of the tail) – Hormonal signs of estrus: Peak serum concentrations of estrogen – Cytologic signs of estrus: Maximum vaginal epithelium cornification Duration of estrus: 7-9 days

32. ESTRUS Cats are known to be “induced ovulators” – Ovulation of the mature follicle occurs as a result of stimulation of the vagina and cervix during mating http://www.youtube.com/watch?v=l6AzSmkoX_g&feature=fvw

33. References Alleice Summers, Common Diseases of Companion Animals http://www.vet.uga.edu/vpp/clerk/Beimborn/in dex.php http://www.vet.uga.edu/vpp/clerk/Beimborn/in dex.php http://veterinarycalendar.dvm360.com/avhc/art icle/articleDetail.jsp?id=727396&pageID=1&s k=&date=