History
A 3-year-old 28.6-kg intact female Labrador Retriever from an intensely managed breeding kennel presented to the Auburn University Small Animal Theriogenology Service from May 7 to 31, 2021, for breeding management and artificial insemination. The historically healthy bitch had 2 uneventful previous litters of puppies, with the last one approximately 6 months prior to presentation, and was current on preventative care.
Upon initial examination, the bitch was bright, alert, and responsive with no abnormal examination findings. The vulva was swollen and discharging serosanguinous fluid. The bitch and sire tested negative for Brucella canis on the Brucella rapid slide agglutination test. Serum progesterone and vaginal cytology samples were taken throughout the estrous cycle (Table 1). Progesterone was analyzed via Immulite 1000 chemiluminescence (Siemens Medical Solutions USA Inc) at the Auburn University Endocrinology Laboratory. Vaginal cytology samples (Figure 1) were taken from the cranial vagina as detailed in previous literature.1
Serum progesterone (ng/mL) and vaginal cytology results of a 3-year-old female Labrador Retriever throughout the estrous cycle starting shortly after the start of serosanguinous vaginal discharge and vulvar edema.
| Date | P4 (ng/mL) | % Cornified | % Anuclear | RBC | WBCd |
|---|---|---|---|---|---|
| 5/07/2021 | < 0.2 | 60a | 10 | 2+ | 1+ |
| 5/10/2021 | 0.2 | 90 | 60 | None | None |
| 5/12/2021 | 0.2 | 100 | 90 | 1+ | Rare |
| 5/14/2021 | 0.6 | 100 | 99 | 1+ | None |
| 5/16/2021 | 0.3 | 100 | 95 | Rare | None |
| 5/18/2021 | 0.3 | 100 | 99 | Rare | None |
| 5/20/2021 | 0.9 | 100 | 80 | Rare | None |
| 5/22/2021 | 1.6 | 100 | 99 | Rare | None |
| 5/23/2021 | 2.2 | 100 | 99 | Rare | None |
| 5/24/2021 | 3.9 | 100 | 50 | Rare | None |
| 5/25/2021 | 7.9 | 100 | 50 | Rare | None |
| 5/26/2021 | 9.1 | 100 | 75 | Rare | None |
| 5/27/2021 | 15.5 | 100 | 80 | Rare | None |
| 5/28/2021 | 18.1 | 100b | 30 | Rare | None |
| 5/29/2021 | 18.6 | 100 | 30 | Rare | None |
| 5/30/2021 | 20.4 | 95 | 10 | Rare | None |
| 5/31/2021 | 18.9 | 40c | 5 | Rare | 3+, Met* |
% Anuclear = Percentage of superficial/cornified cells that are anuclear.
% Cornified = Percentage of epithelial cells on vaginal cytology that are superficial/cornified.
Met* = Metestrum cells present.
P4 = Progesterone.
a–cFigure 1 shows corresponding vaginal cytology photos.
dWBCs are neutrophils predominantly.
Vaginal cytology samples of a 3-year-old female Labrador Retriever throughout the estrous cycle for the purpose of breeding management. A—Proestrus. Approximately 60% of the epithelial cells present are cornified, and a moderate amount of RBCs and minimal amount of WBCs are present. B—Estrus. Approximately 100% of the epithelial cells present are cornified, and rare RBCs and no WBCs are present. C—Diestrus. Approximately 40% of the epithelial cells present are cornified, and rare RBCs and a heavy amount of WBCs with metestrum cells are present. Modified Romanowsky stain; scale bar = 50 µm.
Citation: Journal of the American Veterinary Medical Association 2025; 10.2460/javma.24.11.0726
The bitch was artificially inseminated transcervically on May 27, 2021, with 385 million total, freshly obtained sperm cells (90% progressively motile sperm and 80% morphologically normal sperm). The bitch was bred again via live cover on May 28 and 29, 2021. The owner requested an elective Cesarean section (C-section).
Due Dates
There are 4 peribreeding events that can be used to predict the due date in the bitch: the breeding date(s), luteinizing hormone (LH) surge, ovulation, and cytologic diestrus. Figure 2 summarizes the 4 events and respective calculated due dates in this case.
2021 calendars showing the timeline of events for a 3-year-old female Labrador Retriever through the estrous cycle, breeding parameters, and predicted due dates for the pregnancy. A—May 2021 calendar. The 4 breeding parameter events are denoted on the calendar. B—July to August 2021 calendar. The predicted breeding dates for each of the 4 breeding parameters are denoted on the calendar. Fri = Friday. LH = Luteinizing hormone. Mon = Monday. P4 = Progesterone. Sat = Saturday. Sun = Sunday. Thurs = Thursday. Tues = Tuesday. Wed = Wednesday.
Citation: Journal of the American Veterinary Medical Association 2025; 10.2460/javma.24.11.0726
July 23 to August 7, 2021: 57 to 72 days from breeding date (May 27, 2021)
Breeding dates have the widest range of predicted due dates out of the 4 peribreeding parameters due to the large window in which the bitch is fertile and long survival period of canine semen.1
July 25 to 29, 2021: 65 ± 2 days from LH surge (May 23, 2021)
The LH surge is considered to be the day serum progesterone concentrations reach approximately 2.0 ng/mL. The LH surge can indirectly be determined by measuring serum progesterone in the bitch because progesterone begins to rise prior to ovulation due to the preovulatory luteinization of follicles.
July 25 to 29, 2021: 63 ± 2 days from ovulation (May 25, 2021)
Ovulation is determined to be the day serum progesterone reaches approximately 4 to 10 ng/mL alongside a trend of steadily increasing progesterone over a short period of time. Some practitioners determine ovulation to be when progesterone rises > 3 ng/mL in a 24-hour period and/or when progesterone doubles 48 hours after the LH surge. Ovulation typically occurs 48 to 60 hours after the LH surge.2
July 25 to 29, 2021: 57 ± 2 days from cytologic diestrus (May 31, 2021)
Cytologic diestrus is defined as the day vaginal superficial/cornified cells suddenly decrease by at least 20% and are replaced by small intermediate and parabasal cells.1 Cytologic diestrus typically occurs 5 to 7 days after ovulation and 8 to 9 days after LH surge.1
Treatment and Outcome
On June 25, 2021, pregnancy was confirmed via transabdominal ultrasonography. The bitch returned on July 23, 2021, to begin elective C-section examinations. Based on fetal maturation on transabdominal ultrasound and serum progesterone (Table 2), 7 healthy, fully mature puppies were born via elective C-section on July 25, 2021, without complication.
Serum progesterone and transabdominal ultrasound assessment of fetuses during late-term pregnancy for the purposes of elective Caesarian-section planning of a 3-year-old female Labrador Retriever. Fetal renal development refers to the subjectively determined degree of corticomedullary distinction present.
| Date | P4 (ng/mL) | Transabdominal ultrasound fetal parameters | |||
|---|---|---|---|---|---|
| Heart rates (beats/min) | Renal development | Intestinal development | |||
| Layering | Peristalsis | ||||
| 7/23/2021 | 3.9 | 190–210 | Moderate | Moderate | None |
| 7/24/2021 | 4.47 | 200–240 | Moderate | Moderate | Faint |
| 7/25/2021 | 2.53 | 200–230 | Moderate | Pronounced | Active |
Discussion
Accurately predicting parturition is crucial for C-sections. A significant amount of fetal maturity is not complete until the last few days before parturition. Because of this, incorrect timing for elective C-sections can have devastating consequences when puppies are removed prematurely, as they rarely survive. Additionally, puppies that are removed too late may not survive due to the fetal nutritional demands exceeding the capacity of the placenta within a couple of days.3 Serum progesterone results or vaginal cytology around the time of breeding are extremely valuable in predicting dates of parturition. In 67% of bitches, parturition will occur 63 ± 1 days from ovulation, 65 ± 1 days from the LH surge, or 57 ± 1 days from cytologic diestrus. In 90% of bitches, parturition will occur 63 ± 2 days from ovulation, 65 ± 2 days from the LH surge, or 57 ± 2 days from cytologic diestrus.4
Commonly, serial serum progesterone concentrations alone are used to help assist breeding timing and prediction of parturition dates; however, serial cytology can be used to predict parturition dates as well. In this intensely managed case, both samples were taken for the sake of comparison.
At initial presentation, the bitch appeared to be in proestrus, as evidenced by vulvar edema and serosanguinous vaginal discharge, baseline serum progesterone (below 1.0 ng/mL), and vaginal cytology (< 90% of epithelial cells were superficial/cornified). A proestrus vaginal cytology will have a mixture of parabasal and varying sizes of intermediate epithelial cells.
Three days later, the bitch appeared to be entering cytologic estrus, defined as 90% to 100% cornification of epithelial cells; however, serum progesterone was still baseline. Timing of cytologic estrus varies greatly: typically, anywhere from 6 days prior to the LH surge (or in this case, even earlier) or not until 4 days after the LH surge. Maximum cornification typically lags behind the LH surge by 3 to 6 days and occurs at the time of ovulation 70% of the time.1 Because of this, serial cytologies are not used to determine optimal breeding dates.
The patient was monitored for serum progesterone and vaginal cytology until a rise in progesterone was noticed on May 22, 2021. At this time, progesterone concentration was 1.6 ng/mL and vaginal cytology was consistent with estrus. This is likely the beginning of the LH rise. On May 23, 2021, serum progesterone was 2.2 ng/mL and was considered the LH surge.
We continued to see progesterone rising sharply; on May 24, 2021, progesterone concentration was 3.9 ng/mL; on May 25, it was 7.9 ng/mL; and on May 26, it was 9.1 ng/mL. Given this pattern of progesterone elevation, the patient most likely ovulated on May 25, 2021. On this day, there was a sudden rise within 24 hours. This meets most practitioners’ definition of ovulation: serum progesterone concentration between 4 to 10 ng/mL, with a rise of more than 3 ng/mL over 24 hours or a doubling of progesterone 48 hours after the LH surge. Serum progesterone measurements were continued after suspected ovulation to confirm appropriate cycle progression.
There is a very large window in which a bitch can be bred and still achieve pregnancy. Bitches display behavioral estrus for an average of 9 days, with a wide range of 4 to 24 days. The period of time bitches are receptive to mating does not always line up with this peak fertile window. Sperm can live in the reproductive tract of the female for up to 7 to 10 days, and canine ova can be fertilizable up to 8 to 9 days after ovulation. Ultimately, breeding dates alone are very unreliable in determining when expected parturition will occur.
Cytologic diestrus is defined as the day of a sudden decline of at least 20% of the total superficial cells; more commonly, a drop of over 50% is seen. It is common to see an influx of neutrophils. In this case, cytologic diestrus was observed on May 31, 2021. The occurrence of cytologic diestrus is a reliable method to confirm that ovulation and breeding timing were appropriate and predict parturition. Vaginal cytology is a very simple method to monitor estrous cycles and can be taught to owners to perform at home to prevent daily veterinary visits. It is a useful technique to gain valuable parturition knowledge when dogs are bred via live cover without progesterone timing. Vaginal cytology can be taken on the bitch every day to every other day after breeding until cytologic diestrus is determined.
In this case, fetal maturity was confirmed on transabdominal ultrasound and serum progesterone began to drop to baseline within the predicted window. Bitches begin stage 1 labor within 24 to 36 hours of serum progesterone reaching baseline (below 2 ng/mL). For elective C-sections, surgery should be performed when serum progesterone drops to baseline so surgery can occur before labor begins. In this case, serum progesterone began to reach baseline (2.53 ng/mL) on July 25, 2021. This case demonstrated fetal maturity and serum progesterone dropping in the predicted timeframe for parturition based on LH surge, ovulation, and cytologic diestrus timing.
Ultimately, the large overlap in predicted parturition dates based off of the 4 events demonstrates expected similarity in outcomes and the potentiality of using serum progesterone concentration or vaginal cytology individually for the majority of routine cases. Practically, it is not necessary to calculate parturition from all these parameters.
Acknowledgments
The authors would like to thank Maureen Donohue, LVT, for providing images, Silas Zee for formatting images, and Laura Lee, MA, DVM, DACVP, and Lily Lewis, DVM, for figure assistance.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors have nothing to disclose.
ORCID
J. Klabnik https://orcid.org/0000-0003-4843-6498
References
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Lopate C. Estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review. Theriogenology. 2008;70(3):397-402. doi:10.1016/j.theriogenology.2008.05.034
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Kutzler MA, Mohammed HO, Lamb SV, Meyers-Wallen VN. Accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration. Theriogenology. 2003;60(6):1187-1196. doi:10.1016/S0093-691X(03)00109-2
