Progesterone levels begin to rise at approximately the time of the LH surge (prior to ovulation). Rising progesterone acts
synergistically with declining estrogen to reduce edema of the vulva and vagina, which can be appreciated on vaginoscopic
exam. Other observable clinical signs are minimal. Serial blood samples performed every 2 days may be used to identify the
initial rise in progesterone (usually >2ng/ml) which indicates that the LH surge has occurred. Progesterone can be assayed
by RIA or chemiluminescence at most veterinary commercial laboratories. Several in house semiquantitative kits are also available.
By examination of the range and overlap of progesterone values at different points in female reproductive physiology, it becomes
clear that no one absolute value of progesterone correlates to any particular stage of the cycle. Progesterone varies at the
point of the LH surge from 0.8-3.0ng/ml, from 1.0-8.0ng/ml at ovulation, and from 4.0->20.0ng/ml during the fertile period.
However, if accurate serial quantitative progesterone assays are obtained, the LH surge may be estimated as the day a distinct
increase in progesterone level is seen. While this will not be as accurate as actual identification of the LH surge by use
of an LH assay, estimation by progesterone results is still very useful, and is often more widely available and convenient.
When timing breedings using semi-quantitative in-clinic progesterone assays, only a range of progesterone is obtained, which
makes it difficult to accurately identify the day of the initial rise in progesterone or the true fertile period. Technical
problems with these kits have occurred. Therefore, these assays should only be used for routine breedings where a wider margin
of error is acceptable. A safe rule of thumb to follow is that when testing indicates progesterone has risen above 2 ng/ml,
breeding should begin. Optimal ovulation timing should utilize quantitative progesterone assays from commercial laboratories,
the cost difference is minimal. Regardless of which assay is used, an additional test should always be performed 2-4 days
after the first rise is detected to indicate that the cycle has progressed as expected, a functional corpus luteum has been
formed and ovulation has occurred. When doing progesterone testing for ovulation timing, it is important to remember that
at best, detection of changes in progesterone will give an estimation of the LH surge and the fertile period, and thus is
not as accurate as actual identification of the LH surge with an LH assay.
Breeder clients should be advised to notify the clinic when they first notice a bitch for which timing is planned is in season,
based on vaginal discharge or vulvar swelling/attraction to males. Even the most astute owner can fail to notice the true
onset of proestrus for a few days. Early proestrus should be documented by vaginal cytology (less than 50 % cornification/superficial
cells). A baseline progesterone level might be informative if the true onset of the cycle is unknown (usually 0-1 ng/ml).
Vaginal cytology should be performed every 2-4 days until a significant progression in cornification is seen, usually above
70% superficial cells. At that point, serial hormonal assaying should begin. For routine breedings, progesterone testing may
be done every other day, until a rise in progesterone above 2 ng/ml is identified. The day of the initial rise in progesterone
above 2ng/ml is identified as day "zero". Breedings are advised on days 2, 4 and 6.
When increased accuracy of ovulation timing is necessary (e.g., frozen or chilled semen breedings, infertility cases, breedings
with subfertile stud dogs), daily LH testing is recommended. Once the LH surge is identified, breeding days may be planned.
The day of the LH surge is also day "zero". It is useful to perform vaginal cytology every 2-3 days until full cornification
(>90% superficial cells) occurs. This maximal cornification usually occurs prior to the fertile period is reached and continues
until the onset of diestrus, which is usually a few days after the end of the fertile period. Vaginal cytology may be continued
until the diestral shift is identified, which gives a retrospective evaluation of the breeding just completed. In addition,
at least one progesterone assay should be performed after the LH surge/ initial rise in progesterone is identified to document
that levels continue to rise. This illustrates sustained corpus luteum function and strongly suggests that an ovulatory cycle
has occurred. Extended chilled breedings should occur on days 4 and 6, or 3 and 5 after day "zero". Which two days are chosen
can depend upon overnight shipping possibilities and the involved clients' schedules. Frozen semen breedings should occur
day 5 or 6 after day "zero".
If client economics dictate minimal testing, serum can be batched on a daily basis and quantitative progesterone tests performed
as advised above. When the initial rise in progesterone is identified, the batched serum can be specifically evaluated for
the day of the LH surge, confirming identification of day "zero".
Vaginoscopy may be performed throughout the cycle as an adjunct to vaginal cytology and hormonal assays, especially when evaluating
an unusual cycle. Behavior and other observations should also be made at each examination, but less weight should be put on
these parameters. The clinician should keep in mind that the most accurate ovulating timing occurs when information from several
tests is pooled (vaginal cytologies, vaginoscopy, and progesterone or LH tests).
Ultrasonography may be used to identify ovulation in the bitch. Early attempts were discouraging; the small size of the ovaries
and their similarity to close structures make them difficult to visualize. However, recent reports have identified ovulation
as occurring when a detectable decrease in the number of follicles is seen during serial imaging. The data has shown a close
correlation to the ovulation time established by LH and progesterone levels. While additional research and refinement are
necessary before this method of ovulation timing becomes practical, the results appear promising.
The measurement of glucose in vaginal secretions has been used as a crude guideline for timing breedings by many dog owners.
Increased glucose has been identified in vaginal secretions as an inconsistent finding; it is thought to be a result of insulin
antagonism that occurs due to altered hormone concentrations at the time of the progesterone rise. This finding is not reliable,
however, and so is not recommended for ovulation timing.
Measurement of electrical conductivity of vaginal mucus is used routinely to time breedings in foxes and has been studied
in several other species, including the dog. It was found that electrical resistance increases as estrus approaches and then
plateaus at a maximal level for several days, which has been proposed is a result of rising estrogen levels. While it appeared
that ovulation occurred at some point during this period of maximum electrical resistance, it has not been shown to be correlated
to the LH surge or the fertile period and so cannot be recommended for accurate ovulation timing.