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.
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.
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).
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, relaxin confirms it.
- 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.