Dog Breeding Tests: Progesterone and Relaxin Step by Step
· By Dr. Tang
TL;DR — The working protocol is: start progesterone testing a few days into oestrus, test every 1–2 days until you catch the LH surge (~2–3 ng/mL) and ovulation (~4–10 ng/mL), breed during the 8–15 ng/mL window, then confirm pregnancy with a relaxin test around day 28–30. Two biomarkers, two jobs — progesterone times the breeding, relaxin confirms it — and both need a quantitative number, not a line.
In Plain Terms
Breeding a bitch is a timing problem disguised as a fertility problem. The eggs are only fertilisable for a narrow window after ovulation, and the semen — depending on how it is stored — survives anywhere from 12 hours to 6 days. Get the two windows to overlap and you get a litter; miss by a day and you get an empty cycle. Progesterone is the clock that tells you when the eggs will be ready; relaxin is the confirmation, weeks later, that the whole sequence worked.
The reason this guide exists as its own page is that the failure points are all in the timing, not the fertility. A perfectly healthy bitch and a perfectly good stud can produce nothing if the insemination lands a day too early or too late. The protocol below is the discipline that keeps the timing right.
This is the operational version of what the dog pregnancy test guide explains conceptually. If you want the why, read that. If you want the how — the exact sequence — this is it.
The Protocol at a Glance
| Stage | What you do | What you’re looking for |
|---|---|---|
| Early oestrus | Begin progesterone testing | Baseline (<1 ng/mL) |
| LH surge | Test every 1–2 days | ~2–3 ng/mL |
| Ovulation | Continue daily testing | ~4–10 ng/mL (≈5 typical) |
| Breeding window | Breed / inseminate | 8–15 ng/mL |
| Confirmation | Relaxin test | ~day 28–30 post-ovulation |
The whole thing hangs on one discipline: test early enough and often enough. The rise from 2 ng/mL to 8 ng/mL can happen inside 48 hours, so a test every two days is the minimum cadence, and daily is safer once you see movement.
Step 1: Time Ovulation with Progesterone
Progesterone is the timing instrument. It rises in a predictable pattern around ovulation, and the number maps directly to a decision:
| Progesterone (ng/mL) | What it means | What to do |
|---|---|---|
| < 1 | Too early | Retest in 1–2 days |
| 2–3 | LH surge | Switch to daily testing |
| 4–10 | Ovulation | Note the date — oocytes mature over the next 2–3 days |
| 8–15 | Breeding window | Breed or inseminate now |
| 15–40 | Peak fertility | Late natural mating; consider assisted |
| > 20 | Late | May need TCI or surgical insemination |
The reason this must be quantitative: a strip that says “progesterone present” tells you nothing, because every cycling bitch has progesterone. The decision lives in the number.
For the deeper argument — and the semi-quantitative trap where strips cap at 5 ng/mL, blind exactly where frozen-semen timing lives — see quantitative vs qualitative progesterone.
Step 2: Match Precision to Semen Type
How precisely you need to pin the number depends on what you’re inseminating with:
| Semen | Survival in the bitch | Precision needed |
|---|---|---|
| Fresh | 4–6 days | Coarse — a day early still works |
| Chilled | ~3 days | Moderate |
| Frozen | ~12 hours | Quantitative, 10–20 ng/mL range |
Frozen semen is the case that forces a quantitative reader: it lives about 12 hours, so you need to land insemination in a narrow window that a 5 ng/mL-capped strip can’t see. The stakes aren’t abstract — a 1,400-cycle study found a 1.7-puppy difference per litter (5.6 vs 3.9) between correctly-timed and mistimed frozen-semen breedings.
Step 3: Confirm with Relaxin
Progesterone tells you when to breed; relaxin tells you whether it worked. Relaxin is the pregnancy-only hormone, produced by the placenta after implantation.
| Timing (post-ovulation) | Relaxin status |
|---|---|
| Day 0–20 | Not detectable (no placenta yet) |
| Day 21–28 | First reliable detection |
| Day 28–30 | 95–100% detection — the confirmation window |
| Through term | Stays elevated |
Two practical rules:
- Don’t test too early. Before ~day 21 there’s no placenta and therefore no relaxin — a negative means “too early,” not “not pregnant.”
- One confirmatory test around day 28–30 is usually enough, when levels are comfortably above the detection floor.
For the full relaxin detail — detection windows, false-pregnancy distinction, and the “positive doesn’t always mean viable” caveat — see the relaxin guide.
Why Breeding Fails: The Five Common Errors
Most “failed breedings” are not fertility problems — they are timing errors. The five to diagnose first:
- Testing too late. Start a few days into oestrus, not when you “notice” she’s ready — by then the LH surge (2–3 ng/mL) may already be past.
- Testing too sparsely. The climb from 2 ng/mL to 8 ng/mL fits inside 48 hours. A test every 3–4 days routinely misses the window; every 1–2 days is the minimum.
- Relying on a semi-quantitative strip. A strip that caps at 5 ng/mL is blind exactly where the breeding window (8–15) and frozen-semen timing (10–20) live. It can’t see the number that matters.
- Mistiming frozen semen. Frozen semen survives ~12 hours; landing insemination a day off means a dead straw and an empty cycle.
- Confirming too early. A relaxin test before day 21 reads “negative” because there is no placenta yet — not because she is not pregnant. Test at day 28–30.
Notice that every one of these is a process failure, not a biological one. The fix is not a better stud or a different bitch — it is a tighter testing discipline.
The Full Cycle: From Breeding to Whelping
The protocol does not end at insemination. The same two biomarkers carry you through the whole pregnancy:
| Phase | Marker | What you watch |
|---|---|---|
| Pre-ovulation | Progesterone | The rise: 2–3 → 8–15 ng/mL |
| Insemination | Progesterone | Land inside the breeding window |
| Early pregnancy | Relaxin (day 28–30) | Confirm pregnancy |
| Mid-late pregnancy | Progesterone (optional) | The plateau — should hold >~5 ng/mL |
| Pre-whelping | Progesterone | Falls sharply ~24–48 h before labour |
The mid-late pregnancy step is where serial monitoring earns its keep for at-risk bitches: a corpus luteum that is failing shows a progesterone curve falling toward ~2 ng/mL days before the pregnancy is lost — the window when supplementation can still rescue the litter. For a healthy first-time bitch this step is optional; for a bitch with a history of loss it is the difference between a litter and a resorption. See the pregnancy progesterone curve guide for the full detail.
A Decision Tree for the Busy Breeder
Work through these in order and the protocol runs itself:
- What semen are you using? Fresh (4–6 days) → a coarse window is fine. Chilled (~3 days) → moderate precision. Frozen (~12 hours) → quantitative, no exceptions.
- What is the current progesterone? Below 1 ng/mL → retest in 1–2 days. 2–3 → switch to daily. 4–10 → ovulation noted, oocytes maturing. 8–15 → breed now.
- Is the rise moving fast? A 24-hour jump of 3–4 ng/mL after ~4 ng/mL confirms ovulation and tells you to tighten the cadence.
- Confirm at day 28–30 with relaxin — and only then start planning the whelping box.
What One Cycle Costs in Testing
Plan the program, not the kit. A typical cycle runs:
- 5–8 serial progesterone tests (every 1–2 days from early oestrus through ovulation) — roughly one box of progesterone reagent.
- 1–2 relaxin tests around day 28–30.
Both are 10-test boxes with room-temperature storage and a 2-year shelf life, sold B2B with pricing quoted against volume and terms. A single mistimed frozen-semen insemination costs far more than the entire testing program for that cycle — which is the real economic case for doing the protocol properly.
Related products: Canine Progesterone Test (MG-CA-R020) · Canine Relaxin Test (MG-CA-R021).
A Note on Cats: The Protocol Does Not Transfer
Do not transplant this protocol to cats. Queens are induced ovulators — they do not ovulate on a fixed cycle like bitches; ovulation is triggered by mating itself. That means serial progesterone timing has no feline equivalent, and the “breed at 8–15 ng/mL” concept simply does not apply to a queen. Feline breeding management leans on behavioural signs and vaginal cytology to confirm oestrus, with pregnancy confirmed later by ultrasound or a relaxin test.
The practical rule for anyone breeding both species: keep the two biologies separate. The progesterone number that means “breed now” in a bitch means nothing in a queen — and applying the dog protocol to a cat is how you miss a queen entirely.
FAQ
When should I start progesterone testing?
A few days into oestrus (or when she first accepts the male), then every 1–2 days until ovulation is confirmed.
What level means it’s time to breed?
8–15 ng/mL. LH surge is ~2–3, ovulation ~4–10, and oocytes need 2–3 more days to mature.
When do I confirm pregnancy?
Around day 28–30 post-ovulation, when relaxin is reliably detectable. Before day 21 there’s nothing to detect.
How many tests per cycle?
5–8 serial progesterone tests plus 1–2 relaxin tests — roughly one box of progesterone reagent per cycle.
Does frozen semen change things?
Yes — it survives ~12 hours, so you need to pinpoint 10–20 ng/mL, above the ceiling of semi-quantitative strips.
Key Takeaways
- Two biomarkers, two jobs — progesterone times the breeding (breed at 8–15 ng/mL), relaxin confirms it (reliable from day 28–30).
- Test early and often — the 2→8 ng/mL rise can happen in 48 hours.
- 8–15 ng/mL is the breeding window — a number, not a “positive” line.
- Frozen semen demands quantitative — 10–20 ng/mL is above strip ceilings.
- Confirm at day 28–30 — relaxin is 95–100% detectable by then.
References
- dvm360. “Breeding Management 102 — It’s All in the Timing (Proceedings).”
- Kunanusont N, et al. Vet World. 2021;14(3):585–588. doi:10.14202/vetworld.2021.585-588
- Buff S, et al. (2001). Relaxin detection for early canine pregnancy confirmation (as cited in PMC7126177).
This content is for educational and product-selection purposes only. It is not a substitute for veterinary guidance — work with your veterinarian for insemination timing. Product specifications are as published by Migibio (Guangzhou Magic Biotech Co., Ltd.) and may change.