In reproductive science, dogs are seasonal singly-estrus animals or doubly-estrus animals. [1] Normally, female dogs have estrus twice a year. The sign of estrus in female dogs is vaginal bleeding, which usually lasts for about 21 days. [2] The interval between each estrus is about 6 months. Most dogs kept singly have estrus in spring and autumn (March to May, September to November). According to different sexual physiological changes in dogs, the sexual cycle is divided into four stages: anestrus, proestrus, estrus, and metestrus. [3]
The estrus of French Bulldog is seasonal, usually occurring once in spring and autumn, respectively. A few French Bulldogs have estrus only once a year, while some may have estrus three times. The estrus of French Bulldogs is mainly manifested as vulvar edema, bloody secretions, and acceptance of mounting for mating. The initial estrus of French Bulldogs can occur at 7 to 8 months of age, with an average estrus duration of 9 to 12 days. The mating rate is relatively high during the later period of estrus, meaning that if mating occurs 9 to 12 days after the appearance of bloody secretions, it is highly likely that the dog will become pregnant.
The determination of progesterone and vaginal cytology can be used to determine whether a female dog is in estrus, the stage of estrus, and help determine the optimal time for mating.
1 Experimental Materials
1.1 Experimental Animals
A total of 16 French Bulldog bitches aged 2 to 4 years, in good spirits and appetite, who visited the Tai’aimu Teaching Pet Hospital from October 2017 to April 2018 were selected for the study. The main purpose of the visit was to detect changes in estrus and progesterone levels in dogs.
1.2 Experimental Instruments and Materials
Microscope, progesterone detector, Diff-quik staining kit purchased from Shanghai Naboke Animal Clinical Laboratory, and progesterone detection kit purchased from Beijing Denotech Co., Ltd.
2 Experimental Methods
2.1 Vaginal Smear Observation
2.1.1 Sampling
After the experimental dogs were immobilized, the vaginal area was cleaned, and a sterilized cotton swab soaked in physiological saline was inserted into the vagina for 4 to 6 cm. The swab was gently rotated 2 to 3 times to ensure sufficient contact with the vaginal epithelial cells.
2.1.2 Staining
The vaginal contents collected on the cotton swab were evenly smeared on a glass slide, allowed to air-dry, and stained with Diff-quik. First, the smear to be stained was completely dried and then placed in Fixative (Diff-quik A) for 10 to 20 seconds. Then, the smear was removed from the fixative without rinsing and placed directly into Diff-quik B staining solution for 5 to 10 seconds. The Diff-quik B staining solution should be evenly applied to the slide. Without rinsing, the slide was removed from the Diff-quik B solution and placed directly into Diff-quik C staining solution for 5 to 10 seconds. The Diff-quik C staining solution should also be evenly applied to the slide. After removing the slide, the remaining staining solution was rinsed off with physiological saline, allowed to air-dry, and then examined under a microscope. [4]
2.1.3 Microscopic Observation
Daily records were made of the color and amount of bloody secretions in estrus bitches, as well as the degree of vulvar swelling. Vaginal smears were observed under a microscope, focusing on the morphology, edges, and nuclear membranes of keratinized epithelial cells, and the proportion of keratinized epithelial cells was counted.
2.2 Serum Progesterone Determination
The experimental dogs were immobilized, shaved, and disinfected. Then, 2 ml of blood was collected from the cephalic vein, centrifuged at 12000 r/min for 3 minutes to separate the serum. The serum was extracted using a pipette, and 50 μl of serum was mixed with diluent. Then, 100 μl of the mixed solution was added to the sample well of the reagent card. Once the mixed solution flowed from the round hole to the front space, the reagent card was inserted into the slot of the fluorometer. The “Start Test” button on the fluorometer screen was clicked, and the test results were read after 10 minutes.