Progesterone Timing

By William E. Schultz, D.V.M.

Progesterone is one indicator of ovulation in the bitch. Progesterone is secreted from the ovaries and is the hormone that maintains pregnancy. Progesterone is present in very small amounts before ovulation. Just before ovulation the progesterone rises above a baseline to greater than 2 nanograms and continues during the pregnancy phase of a dog’s heat cycle. This phase is called diestrus. Whether or not a bitch is pregnant the progesterone rises and stays elevated during the diestral phase. Progesterone declines rapidly just before the bitch goes into labor.

Prior to progesterone testing vaginal cytology was the only method used to set a breeding date. This method worked fairly well, but has the potential to be quite a bit off in its prognosticating ability. Most bitches cornify around day 8 to 9 and are ready to be bred by day 11 to day 14. Some bitches will completely cornify by day 3 or 4 of their cycle causing early breeding and lack of pregnancy. (Cornification is the superficial cellular change which involves thickening of the vagina to prevent damage to the bitch during natural breeding. Between heat cycles, the wall of the vagina is very thin and it becomes thickened during the early phases of the heat cycle. When a swab is taken of the cells on the surface of the vagina, the changes which occur are indicators of the stage of the heat cycle. The superficial cells start out as very small cells with large nuclei. As the cycle progresses, these cells become much larger with a much smaller nucleus and the edges begin to deteriorate. These mature cells are "cornified" and the percentage of these cells approaches 100% as the bitch nears standing heat). Progesterone testing is dramatically more accurate in determining ovulation and which day to inseminate.

When progesterone testing first arrived we had several different companies making in-house semi-quantitative tests. These tests require interpretation of a blue color that changes with a rise in progesterone. These are still used with moderate success when quantitative progesterone is not available.

Currently, almost all regional labs and universities will run quantitative progesterone. The results are given in nanograms or nanaomols. It is important to know which lab did the testing because most veterinarians use the results in nanograms. A nanogram is roughly 1/3 of a nanamol. Canada and Michigan State University give results in nanomols and the results must be converted to nanograms to avoid misunderstandings in timing.

Blood taken for progesterone testing must be handled with care. We do not use vaccutainers for the blood draw because of damage to red blood cells during the collection. A 20-gauge needle and a 6 ml syringe are used for sample collection from the jugular vein, if possible, and the blood is then placed in a red top vaccutainer. The top is removed from the vaccutainer and the needle is removed from the syringe allowing the blood to be placed in the tube with the least trauma. Break down of red blood cells or hemolysis will alter the results. The tube is placed in a test tube rack and a timer is set for 9 minutes. The tube is then centrifuged with another timer running and the serum is removed and stored in the refrigerator while waiting to be taken or sent to the lab. If your veterinarian is not following a similar protocol your testing will be inaccurate.

Progesterone testing is usually done every other day and, in the early days of the cycle, may be done every 3 to 4 days. It is important to know that if the rise in progesterone is missed, guesswork is needed to determine the actual day for the breeding. We determine breeding days for natural or transcervical breeding at 3 and 5 days after the progesterone rises above 2 nanograms. We do a confidence test one to two days later to confirm a rise above 5 nanograms. The rise in progesterone at 2 nanograms is indicative of the preovulatory LH (leuteotropic hormone) surge. This is followed two days later by ovulation. If the progesterone rises slowly we will delay breeding accordingly. For frozen semen breedings we will do LH testing at the time of the initial rise in progesterone for the most accurate prediction of ovulation. This is not usually needed for a fresh or fresh chilled breeding because of the longevity of the semen. Fresh and fresh chilled semen may live longer than 4 days while frozen semen, once thawed, will only live 12 to 18 hours necessitating the more accurate timing.

For more information on Progesterone Timing, please download our paper by clicking here.