This section explains the science and tests behind both vaginal cytology and measurement of blood progesterone levels

 

Vaginal cytology

 

The vaginal mucosa is a target organ for oestrogen and changes during oestrus from a two to four-layered and then a multilayered epithelium.  This is observed on microscopy as an increasing percentage of keratinized (cornified) epithelial cells.  The phase of maximum keratinization occurs at least 3-6 days after the peak of oestrogen secretion and is therefore close to the phase of ovulation (stimulated by the luteinising hormone [LH] surge) (see fig. iii)  Although deep vaginal sampling is more grossly remarkable cells removed from the vestibular vaginal area are sufficient to demonstrate changes.

 

Fig. ii.  Vaginal cytology identification (diagram & plates) and method

 

1. Cells are removed by gently scraping the vestibular mucosa with a glass rod, wooden spatula or small curette.  Alternatively, a glass pipette can be used to remove vaginal fluid.

 

2. Transfer to a grease free, clean glass slide.

 

Note: The same removal method and site should be selected for repeat tests.

 

3. Stain using Modified Schorr's trichrome stain or Leishman's stain.

 

 

 

 

 

 

 

 

 

 

 

 

 

Proestrus                                                                      Late Poestrus

 

 

 

 

 

 

 

 

 

 

 

 

Oestrus                                                                          Dioestrus

 

 

For Hormonal changes during the bitches heat or season also see mating your bitch part one.

 

The concentration of oestrogen secreted by the maturing ovarian follicle is reduced as the peak of Luteal (luteinizing hormone) activity is reached.  Ovulation normally happens 48 hours after the peak of LH.

 

Progesterone is produced by the follicles just before the rise in LH.  Around the time of ovulation the concentration of blood progesterone progressively rises and at ovulation the level of progesterone is generally between 4 and 10 ng/ml.  With the "average bitch" this happens 11-13 days after the start of pro-oestrus.  However, because of the large variations between breeds and individuals one can only be sure that ovulation has taken place when the level of progesterone has exceeded 10ng/ml.

 

The ova (eggs) then take two to three days to mature. The bitch should be mated in the 24-48 hours which follow the 10ng/ml level of progesterone, repeating possibly 48 hours later.

 

Because the spermatozoa can survive for relatively long periods (up to a week in some cases) in the genital tract of the bitch fertile matings resulting in one or two puppies have occurred up after a mating up to nine days prior to and six days after ovulation (England 1996).  Litter sizes are largest, however, when matings take place with four days either side of ovulation.

 

 

Measurement of blood progesterone

 

This can be done quantitatively through some laboratories or alternatively using a commercial test Ovucheck Premate which most veterinary practices are able to perform on site.  This is a semiquantitative test to measure the progesterone concentration in a sample of plasma or serum.  The result is determined by comparing the colour of the treated sample against the colour of high and low progesterone standards.  The low standard concentration (A) is equivalent to 3 ng/ml and the high standard (B) 10 ng/ml of blood progesterone.

 

To check the validity of the test, the operator must ensure that the low standard is a darker shade of pink than the high standard (the lighter colour indicates a higher level of progesterone)

 

The test sample is then compared with each of the standards:

 

If the test sample is as pink or more pink than the low standard this indicates it has a concentration of 3 ng/ml or less of progesterone.  The bitch is therefore still in pro-oestrus and should be tested again in two days time.

 

If the test sample is clearly a lighter pink than the low standard, but darker than the high standard, the level of progesterone is between 3ng/ml and 10 ng/ml.  This indicates that oestrus has probably started but ovulation may not have occurred.

If the test sample has the same colour as B (high standard) the level of progesterone has reached 10ng/ml.  This indicates that ovulation has probably taken place.  A period of 2 to 3 days is then necessary for the ovum to mature.  Therefore mating should take place within the next 24 to 48 hours possibly repeating in 24 to 48 hours.

 

If the test sample is lighter than B (high standard) the level of progesterone has exceeded 10ng/ml.  This is a strong indication that ovulation has already taken place.  How long before will depend upon previous test history.  Mating should take place within 2 to 9 hours after this result possibly repeating 8 hours later.

 

Note the test is temperature sensitive and if the environmental temperature is lower than 15 to 20 C it may be necessary to prolong the incubation periods.