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Friday, March 27, 2015

Tracking the canine estrous cycle

Keywords: canine, estrous, cycle, LH, ovulation, breeding

The author devised the first and second images for teaching and to guide clients interested in canine reproduction. Please note that they have high resolution (300 dpi ); suitable for printing and distribution to students and clients.

Image size: 3129 x 2568 px (300 dpi. suitable for printing)

This is an important image to bear in mind when handling either normal or abnormal canine reproduction. Of special note is the association between the LH surge and the first rise in serum progesterone (P4) and the time when the oocytes first become fertile. This is important because LH is more complex and more expensive to measure than P4. Also, serial LH measurements even at 24 hour intervals can fail to detect the LH surge, spanning it completely in some bitches.

The image below serves as a key for the larger image. It features are discussed below.

Image size: 1145 x 606 px (300 dpi. suitable for printing)

Notes on the key image:
In bitches, proestrus (characterized by proestral bleeding, the onset of vulvar swelling and attraction of males) may be as brief as 2 or 3 days or as long as 30 days (usually longer in younger bitches). Also, the period during which a bitch will allow mating is highly variable as well; older bitches may accept male earlier, young bitches later. The successively darker silhouettes of  copulating dogs in the image indicates the likelihood with which a bitch will accept breeding. Some bitches will never accept males.  In essence, it is unreliable to base breeding decisions on the onset or duration of proestrus.

Spermatozoa can remain fertile in the reproductive tract of bitches for up to 11 days. Therefore pregnancies can result even when bitches are bred long before their oocytes become fertilizable. Although the possible time span of ovulations is not known in practice, data suggest that ovulation may continue for several days in some bitches. Therefore fertilization can occur over several days as well, even though the oocytes from earlier ovulation have already begun to degenerate (oocytes begin to degenerate within 48 hours after the formation of the first polar body). Combined with the longevity of sperm in the tract, the spread in availability of fertilizable oocytes can provide pregnancies under the most mismanaged of breedings. 

During early proestrus, ovarian follicle are growing and begin to produce significant amounts of estrogen. Estrogen brings about changes in behavioral centers in the brain, induces the formation of pheromones such as methyl p-hydroxybenzoate (MPHB) and hemorrhage via diapidesis from the endometrium. In addition, and importantly from a clinical standpoint, estrogen causes sodium retention in target tissues and consequently, water retention as well. This causes edema of the vulvar lips and vaginal epithelium. It is only when serum estrogen concentrations drop (at about the time that the LH concentrations are beginning to surge) that vulvar and vaginal edema suddenly decreases, allowing copulation to occur comfortably.

In inset A of the endoscopic views below, the vaginal mucosa is edematous. The external urethral orifice is at the center of this image. This image was obtained from a bitch during proestrus. Hemorrhage was indeed present but lay within the folds seen in this image. Inset  B is a view of the vagina from a bitch that had ovulated recently. Edema had mostly disappeared and the vaginal epithelium had taken on a "crinkled" or "notched" appearance. "Crena" is a Latin term denoting a notch (as in the edge of a leaf) and notching is referred to as "crenulation". It is also the accepted term for this appearance in the vagina. Crenulation is one of several markers of the onset of the fertile period in bitches. Image B was obtained from one of many bitches that still show endometrial hemorrhage at the time of the LH surge.

Image size: 500 x 261px

Because the LH surge occurs at about the same time as a sudden decrease in vulvar tone, vulva tone can be used successfully to determine the optimal time for breeding. It is probably not as precise as using LH or P4 monitoring. That remains to be tested, but the author is aware of at least one Beagle breeding colony where it is used routinely to determine optimal times for breeding. When frozen semen is used, it is probably safer to determine breeding times by monitoring  LH or P4 concentrations until data shows otherwise.

The mitogenic effect of estrogens causes parabasal cells in the vaginal epithelium to divide. This pushes the more superficial cells away from the basement membrane (their source of oxygen and nutrition) causing them to die. Their nuclei undergo pyknosis and karyorrhexis and are finally lost altogether. This provides the vaginal cytology associated with estrus. After ovulation and the decline of serum estrogen concentrations, the process is reversed. Changes in vaginal cytology are valuable to determine if a bitch is sufficiently advanced in proestrus for P4 or LH sampling to begin. However, cytology alone is not sufficiently accurate to be used to determine the optimal time for breeding.

The LH surge lasts for 24 to 48 hours and brings about maturation and ovulation of the follicles that have been growing during proestrus.  These follicles begin to ovulate about two days after the the LH surge and as mentioned earlier, ovulation probably continues over a few days. Most domestic animals ovulate oocytes that have already formed the first polar body at the time of ovulation and only await sperm penetration to stimulate the completion of meiosis i.e the second (meiotic) division of meiosis. However, the reduction division of meiosis has not yet occurred when bitches ovulate. That process will take another two to three days to occur i.e. canine oocytes can not be fertilized at the time of ovulation. Indeed, bitches are at their most fertile only four to five days after the LH surge. Interestingly, bitches are sexually receptive at this time although serum P4 concentrations are rising rapidly. This is very different to the situation in other domestic animals.

Together with a few other animals (including humans) luteinization of follicles actually begins before ovulation. Due to this physiological quirk, it is possible to monitor serum P4 concentrations to determine the approximate time of the LH surge.  When serum P4 concentrations first exceed 2 ng/mL (~ 6.5 nmol/L) it is highly probable that the LH surge is occurring. This means that a bitch should be bred four to five days later if a single breeding is to be done. If several breeding are to occur, they should span the approximate period from 3 to 7 days after the LH surge.

As the main image shows, owners can be misled into believing the a bitch has had a gestation as short as 56 days or as long as 72 days from the time of breeding. In fact, the gestation length of dogs is fairly consistent at about 6o days. It is often stated that gestation is 65 days + in length after the LH surge but of course that is not true because gestation only begins after fertilization. Pregnancies that are shorter that 60 days probably result from ovulations that occur several days after the LH surge.

Once the day of the LH surge is known, one knows exactly when to breed a bitch naturally or with fresh or frozen-thawed semen. The day of whelping after that time will be very close to 60 days. This allows one to schedule pregnancy diagnoses and to plan for whelping. It also allows one to determine the optimal time for an elective cesarean sections should that be required.


Concannon, P.W. 2012. Research Challenges in Endocrine Aspects of Canine Ovarian Cycles. Reprod Dom Anim 47 (Suppl. 6), 6–12

Saint-Dizier. M et al. 2001. Induction of final maturation by sperm penetration in canine oocytes. Reproduction 121: 97–105

England, G.C.W et al.2006 Relationship between the fertile period and sperm transport in the bitch. Theriogenology. 66:1410-1418

Hori,T. et al. 2012. Ovulation Day After Onset of Vulval Bleeding in a Beagle Colony.
Reprod Dom Anim 47 (Suppl. 6), 47–51

Thomassen, R. et al. 2006. Artificial insemination with frozen semen in dogs: A retrospective study of 10 years using a non-surgical approach. Theriogenology. 66:1645–1650