The correct method for testing progesterone levels in dogs mainly involves the following steps:

Sample Collection:
Sample Type: Serum or plasma is typically used as the sample for testing.
Sample Collection Method: Obtain blood samples from the dog through venous blood collection.
Sample Processing: Centrifuge the collected blood samples to separate the serum or plasma, carefully collect the supernatant, and store it.

Testing Preparation:
Reagents and Equipment: Include progesterone ELISA kit, microplate reader (450nm wavelength), precision pipettes and tips, 37°C incubator, etc.
Sample Dilution: If the progesterone concentration in the sample is too high, appropriate dilution may be required.

Experimental Procedure:
Prepare the microplate coated with progesterone capture antibodies according to the instructions of the progesterone ELISA kit.
Add the sample, detection antibody, and substrate TMB to the wells sequentially to form an antibody-antigen-enzyme-labeled antibody complex.
After washing, add the substrate TMB to develop color, which will turn yellow under the action of the enzyme.
Measure the optical density (OD value) at 450nm using a microplate reader.

Result Calculation:
Calculate the progesterone content in the sample based on the standard curve using the OD value.
The progesterone index is usually expressed in ng/ml.

Testing Time and Frequency:
Initial Testing Time: Perform the first progesterone test on the 7th day after the onset of estrus in female dogs.
Testing Frequency: Test every 24-48 hours, preferably every 24 hours.
End of Testing: Stop testing after determining the appropriate breeding time.

Notes:
Progesterone testing should be performed under the guidance of a professional veterinarian to ensure accurate operation.
During the testing process, attention should be paid to maintaining the stability of samples and reagents to avoid contamination and errors.
The results of progesterone testing may be affected by various factors such as individual differences and disease status, so it is necessary to make comprehensive judgments based on other clinical information.

Through the above steps, progesterone levels in dogs can be accurately tested, providing strong support for dog breeding and health management.