Monash Veterinary Clinic

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Techniques for Timing of Inseminations

Vaginal Smears (Cytology)

Vaginal smears are one of the most useful tools for assessment of the stage of cycle of the bitch, however they are the most underutilized test by most Veterinarians. The test is both simple and cheap, however confidence in interpretation is often lacking. 

Often the wrong significance is placed on the need for vaginal smears. Too often vaginal smears are taken from a bitch and sent to the laboratory for microbacterial culture and sensitivity testing to see if the bitch ‘has an infection’ prior to mating. It must be remembered that the vagina of the bitch is not a sterile environment; it is a body cavity open to the outside world, just like the rectum and oral cavity. To culture the vagina is not an accurate test for the presence of infection. The most important aspect of vaginal smears during the ‘heat’ period is to assess the stage and progression of the cycle.

There is a stage early in the season and late in the season where the vaginal smears look very similar. Vaginal smears are not accurate to define ovulation or the fertile period, their use is to help stage the position in the heat cycle and to follow the normal progression of the cycle. They are very helpful in defining split-heats and alongside high progesterone readings as to whether the bitch is still in the fertile period.

Progesterone Assays

Progesterone is a hormone released by the ovary which indirectly indicates what is happening with the oocytes (eggs) from the ovary.

Progesterone assays are an important aspect in timing for mating and artificial insemination. It is important to be consistent in both the collection (technique and handling) and the laboratory used in the determination of progesterone values.

The two most commonly used methods for Progesterone determination by laboratories are Chemoluminesence and Radioimmunoassay. Chemoluminescence is the more favoured test by Theriogenologists world wide. Monash Veterinary Clinic uses Healthscope for all of its blood progesterone assays. In house test kits, for example date to mate tests , are a semiquantitative test measuring progesterone via an ELISA; they are useful as a guide only for progesterone levels and are not as accurate as laboratory assays.

As with all hormone assays results will vary between laboratories greatly due to the following reasons: differing technique of measurement, different suppliers of reagents for the same technique, different machine manufacturers. Therefore it is important to have the assays for the bitches season to be done at the same laboratory to avoid confusion. We are looking for changes in values in conjunction with behavioural, cytological and vaginoscopic changes to pinpoint our location within the cycle.

Progesterone has been shown to be higher in the morning compared to the evening in pregnant bitches. No correlation has yet to be shown in the non pregnant bitch during her season. It is recommended to be taking the samples at similar times of day each time to look for the change in progesterone levels.

Blood for progesterone measurement should be collected into plain tubes with no gel or lithium heparin tubes. The gel in clot activator tubes has been shown to affect progesterone values in human testing. Red blood cells will absorb progesterone from the sample, and as a rule of thumb if the sample is not to be analysed for more than 4 hours by the laboratory then the sample should be spun and the serum sent to the laboratory. Once separated, the progesterone will be stable in the serum for many days. Refrigeration of the whole blood sample initially (the first two hours) will also affect the progesterone level of the sample. We recommend collecting into a red top plain tube with no gel, not refrigerating the sample, and spinning it down if the sample is not to be analysed within 2 hours.

As with most blood tests, the animal is recommended to be fasted before sample collection. Eating will increase the liver metabolism of progesterone and affect results.


Vaginoscopy is an essential tool and skill required for good timing of inseminations. It helps to confirm what the progesterone and vaginal cytology results indicate. Vaginoscopy is imperative in the assessment of the heat cycle in which frozen thawed semen will be used.

Vaginoscopy is performed at the level of the posterior and mid vagina, using a rigid endoscope or sigmoidoscope.