The average bitch will experience the LH surge on Day 10 (where Day 1 is defined as the first day that bloody discharge is observed), will ovulate on Day 12, and will, therefore, optimally conceive on Day 14.

Traditionally, bitches were usually bred on the 14th day following onset of proestrus. This was because it was observed that most bitches would display "tail flagging", defined as the lateral deviation of the tail with elevation of the vulva, and "standing heat", defined as the bitches' behavior in allowing the male to mount and breed, at this point in time. Additionally, other physical changes such as a softening of the flesh of the swollen vulva were external signs indicating the onset of ovulation. Later, when it became the norm to perform multiple matings, the bitch was bred on Days 12 and 14 (for double service), or Days 11, 13, and 15 (for triple service). Though these schedules are still adequate for insuring optimal breeding and litter size in the average bitch, not all bitches ovulate on Day 12 following proestrus onset. Some may ovulate as early as Day 5 or as late as Day 25 in which case utilizing this standard mating schedule will result in breeding failure.

Using receptive behavior of the female as an indicator for ovulation and therefore, a method for determining mating schedule has many limitations because these approaches are not always clear-cut. Some bitches may exhibit "phantom proestrus" (displaying little or no outward signs of bloody discharge, etc.) making it difficult to estimate the average date of ovulation, may "flag" and appear receptive to males throughout proestrus, or may remain unwilling to mate even following ovulation. The differences observed from bitch to bitch in regard to mating signs and behavior as well as the fact that unsuccessful mating will result in a 6-month or longer wait to "try again" understandably leads to a sense of anxiety in many bitch owners. Additionally, even cytological analysis, which can be used to determine onset of estrus, is often a poor predictor for ovulation since LH surge, a key precursor to ovulation, may occur 3 to 5 days before to 5 days after the onset of estrus. Therefore, a clinical screening test that accurately predicts ovulation in the bitch and therefore serves to optimize breeding schedule, is used frequently by many breeders.

The serum progesterone Enzyme-Linked ImmunoSorbent Assay (ELISA) is an accurate predictor for ovulation.

For this assay, vaginal smears are examined periodically at the onset of proestrus to monitor cornification of the vaginal epithelial cells, which occurs as a result of increasing estrogen hormone. When the cells of the vaginal wall are approximately 60% cornified, as observed by microscopic analysis, testing with the serum-progesterone ELISA should commence. Blood samples are drawn, ideally, every day (though every 2 days may also be utilized), and whole blood or serum (depending on the test kit utilized) is added to a test indicator that has been treated with monoclonal antibodies specific for progesterone. Late in proestrus, the level of estrogen will decrease and levels of LH will surge. This LH surge is concurrent with an increase in progesterone levels, which will rise above 1 ng/ml on the same day. Therefore, detection of increased serum progesterone corresponds to the LH surge. This is an important indicator, since ovulation occurs 2 days following the LH surge. The serum progesterone ELISA manufactured by International Canine Genetics, a division of Synbiotics (Malvern, PA) provides a qualitative color change to indicate when the progesterone level in the bitches' serum rises. Early in proestrus, the test indicator will produce a strong blue color indicating low levels of progesterone (between 0.0 to 1.0 ng/ml). For determination of LH surge, the first appearance of a fading of the test color (as compared to an internal "low-progesterone" control indicator) to a light blue (approximately 2.0 ng/ml) indicates the LH surge. Two days later upon ovulation, progesterone levels will further increase to 5.0 ng/ml or above, at which point the test color will appear white, confirming ovulation. Though different kits utilize different testing methods and procedures, the concept of each kit is virtually identical and provides the means for determining ovulation. However, limitations to the sensitivity of ELISA testing may sometimes result in false-positive and false-negative results. This is because the greatest inaccuracy in measuring serum progesterone levels occurs in the range of 1.5 to 3.0 ng/ml of progesterone, the concentration range of importance for determining the LH surge. Greater accuracy occurs in the high range of greater than 5.0 ng/ml. Occasionally, a test will indicate a "medium" level of progesterone one day (suggesting LH surge), but may indicate a "low" level of progesterone when taken on the next day. This suggests that the former test demonstrated a false-positive because once progesterone levels rise, they should remain elevated and increase throughout ovulation. Therefore, to reduce incidence of inoptimal mating due to false-positives, two consecutive days of testing, whereby increased progesterone levels are indicated on both days, should be obtained prior to establishing the mating schedule. Additionally, a post-ovulatory testing should be performed on a day that mating is performed to confirm high levels of progesterone (5.0 ng/ml or greater), which indicates that ovulation has occurred.

Alternatively, ICG also offers an LH ELISA that works on the same principle as the progesterone ELISA but which specifically detects levels of serum LH. Though the LH ELISA may be used alone for determining ovulation, the major limitation with the LH ELISA occurs as a result of the brief time in which the LH concentration is elevated in the serum. Unlike progesterone concentration, which continues to increase, LH peaks within 24 hours and then quickly dissipates. As such, it is possible to miss the LH surge if one does not test on a consistent, daily basis. For this reason, Synbiotics recommends combined testing with the progesterone ELISA and the LH ELISA, using the latter to reduce the possibility of false-positives and false-negatives occasionally encountered with the progesterone ELISA. For example, on the first day that the progesterone ELISA indicates a rise in serum progesterone levels, one may confirm the concurrent LH surge by re-testing serum using the LH ELISA. If both tests are positive, then there is less likelihood of false-positive results.

Once the day of the LH surge is determined (to be considered Day 0), ovulation will occur on Day 2. Maximal litter size is achieved when the bitch is bred 2 days after ovulation (Day 4 following the LH surge). A single insemination 2 to 3 days following ovulation will result in pregnancy in the healthy reproductive bitch. The reason that optimal conception occurs 2 days following ovulation is because when ovulation occurs, the ova are immature (primary oocytes) and must undergo two meiotic divisions before they can be fertilized. These divisions can take up to 48 to 72 hours to occur. Once matured, the ova remain viable for another 2 to 3 days. Because normal sperm (spermatozoa) of the male delivered by natural insemination can live in the reproductive tract for at least 5 to 6 days, successful conception may occur if a bitch is bred from 2 days prior to ovulation to 4 days after ovulation.