Dog Pregnancy Tests: Relaxin and Progesterone Explained
· By Dr. Tang
TL;DR — Two hormones, two jobs. Progesterone tells you when to breed: it climbs from <1 ng/mL through a 2–3 ng/mL LH surge, ~5 ng/mL ovulation, and an 8–15 ng/mL optimal window. Relaxin tells you whether it worked: it’s the only pregnancy-only hormone and is reliably detectable from ~day 21–28. A strip that just says “progesterone present” is useless — every cycling bitch has progesterone. And a semi-quantitative strip capped at 5 ng/mL is blind precisely where frozen-semen timing (10–20 ng/mL) lives.
Most confusion in dog breeding comes from treating progesterone and relaxin as interchangeable “pregnancy hormones.” They’re not. One is a timing instrument, the other is a confirmation instrument, and confusing them costs litters.
The Two Hormones People Keep Confusing
Here’s the split in one paragraph, because it resolves about 80% of the wrong-test problems we see from breeders:
- Progesterone is produced by the ovary during every oestrous cycle — pregnant or not. It rises through proestrus, peaks around ovulation, and stays high through diestrus. That means it is superb for locating ovulation in time, and useless for confirming pregnancy. A “high progesterone” result two weeks after mating tells you the dog cycled. It does not tell you she conceived.
- Relaxin is produced by the placenta — and only the placenta. No placenta, no relaxin. It first appears after implantation and climbs steadily through gestation. That makes it the definitive blood marker for “yes, she’s actually pregnant.”
If you remember one thing, remember this: progesterone times the breeding, relaxin confirms the pregnancy. Using one for the other’s job is how breeders end up either breeding blind or celebrating a false positive.
Progesterone: The Timing Hormone
Why “Positive” Is a Useless Answer
A qualitative strip that returns “progesterone present” answers a question nobody asked. Every intact bitch in oestrus has progesterone. The value you need is how much, because the quantity maps directly onto a decision:
| Progesterone (ng/mL) | What it means | What to do |
|---|---|---|
| < 1 | Anoestrus / too early | Wait, retest in 1–2 days |
| 2–3 | LH surge | Increase testing frequency to daily |
| 4–10 | Ovulation (~5 typical) | Note the date; canine oocytes need 2–3 more days to mature |
| 8–15 | Optimal breeding window | Breed / inseminate now |
| 15–40 | Peak fertility | Late natural mating; consider assisted |
| > 20 | Late | May need transcervical (TCI) or surgical insemination |
The gap between 2 ng/mL and 8 ng/mL is the gap between “not yet” and “breed now” — and it can close inside 48 hours. That’s the entire argument for a quantitative number over a yes/no line.
The Semi-Quantitative Trap (This Is the Part Most Guides Skip)
Between a pure yes/no strip and a full quantitative analyzer sits the “semi-quantitative” kit — a visual band that brackets progesterone into a few ranges. Here’s the problem nobody advertises:
Semi-quantitative progesterone strips typically cap around 5 ng/mL. For a natural mating with fresh semen that’s borderline workable, because fresh semen survives 4–6 days in the bitch and you have slack. But for frozen semen — which survives roughly 12 hours — you need to pin down 10–20 ng/mL, and a strip that maxes out at 5 can’t see that region at all. It’s not a small accuracy gap; it’s a structural blind spot exactly where the stakes are highest.
There’s real data behind why this matters. A study across 1,400+ breeding cycles found that bitches with a slow progesterone rise inseminated with frozen semen produced 3.9 puppies on average, versus 5.6 for a normal (“fast”) rise with correct timing. That’s a 1.7-puppy difference per litter — driven almost entirely by whether the progesterone number was read precisely enough to time insemination to the short frozen-semen window.
The takeaway: if you breed with fresh semen you can tolerate a coarse test. The moment frozen or chilled semen enters the picture, you need a quantitative number, and a 5 ng/mL ceiling is no longer acceptable.
Relaxin: The Confirmation Hormone
Relaxin’s value is that it’s unambiguous. It is secreted by the developing placenta after implantation, so a positive relaxin result means placental tissue exists — which means pregnancy.
The Detection Timeline
| Timing (post-ovulation) | Relaxin status |
|---|---|
| Day 0–20 | Not detectable (no placenta yet) |
| ~Day 21–28 | First reliable detection (earliest ~25 days in some dogs) |
| Day 30 | 95–100% detection rate |
| Through term | Remains elevated until parturition |
Two practical consequences fall out of this table:
- You cannot test earlier than ~3 weeks. Before implantation there is no placenta and therefore no relaxin. A negative result on day 15 means nothing — it’s a “too early,” not a “not pregnant.”
- Relaxin does not “disappear after two weeks.” That’s a myth we see repeated in old guides. Relaxin stays elevated for the entire pregnancy. What is true is that the best window for a single confirmatory test is day 28–35, when levels are comfortably above the detection floor and a single draw gives you a clear answer.
How These Tests Actually Work
Both progesterone and relaxin kits on Migibio’s platform use fluorescence immunochromatography (FICT) rather than the colloidal-gold strips people picture. The difference matters for two reasons:
- It’s quantitative. Fluorescent intensity is directly proportional to analyte concentration, so the reader returns a number (ng/mL, pg/mL) — not a subjective “how dark is the line.” This is what makes the cut-off table above usable.
- It’s read by an instrument, not by eye. Sensitivity reaches pg/mL level with a CV (coefficient of variation) under 10%, so the number you get at 2 ng/mL is as trustworthy as the one at 15 ng/mL.
On the hardware side, breeders and clinics have two choices:
| FIA680 | FIA880 | |
|---|---|---|
| Channels | 1 (single) | 6 (parallel) |
| Result time | 3–11 min | 3–15 min |
| Throughput | Up to 200 tests/hour | Up to 300 tests/hour |
| Temperature control | Standard | Constant-temperature |
| Best for | Small clinics, breeders, mobile vets | Hospitals, reference labs, multi-user |
For a breeder running serial progesterone curves on one bitch, a single-channel FIA680 is the economical choice. For a busy clinic running progesterone, thyroid and cardiac panels across several rooms at once, the FIA880’s six channels mean nobody queues.
What a Breeder Actually Needs to Buy
This is where the “test” and the “testing program” part ways. A single progesterone number is nearly worthless; a series is what times a breeding. So budget for the program, not the kit.
The reagents:
- cProgesterone (cat. MG-CA-R020) — for ovulation timing
- cRelaxin (cat. MG-CA-R021) — for pregnancy confirmation
Both are 10-test boxes, store at room temperature, and carry a 2-year shelf life.
The rough math on one cycle:
A typical breeding cycle runs 5–8 serial progesterone tests — you start a few days into oestrus and test every 1–2 days until the surge and ovulation are confirmed, then run one or two relaxin tests around day 28–30 to confirm. So plan for roughly one box of progesterone reagent per cycle, plus a relaxin test or two. It’s a small line item next to the cost of a missed window — a single mistimed frozen-semen insemination wastes more than the entire testing program for that cycle.
One honest note for anyone coming from retail pet sites: Migibio is a manufacturer, and these kits are sold B2B — typically to veterinary clinics, distributors and professional breeders, with a minimum order around 10 boxes. We don’t publish a fixed per-box retail price for the same reason your clinic’s supplier doesn’t put one on a public page: it depends on volume, region and OEM/private-label terms. If you’re a breeder buying for one dog, the practical route is through your vet or a distributor; if you’re a clinic or importer running volume, the number you care about is the per-test landed cost at your MOQ, which is a quote, not a list price.
Frozen Semen Changes the Math
Everything above about “semi-quantitative is fine for fresh semen” collapses the moment you freeze. Put the numbers side by side and the risk becomes obvious:
| Fresh semen | Chilled | Frozen | |
|---|---|---|---|
| Sperm survival in the bitch | 4–6 days | ~3 days | ~12 hours |
| Timing tolerance | Wide — breed a day “early” and it still works | Moderate | Near-zero — hours matter |
| Progesterone precision needed | Coarse (strip acceptable) | Moderate | Quantitative, 10–20 ng/mL range |
Frozen semen lives about 12 hours. A semi-quantitative strip capped at 5 ng/mL cannot see the 10–20 ng/mL region where frozen-semen insemination must land — which is why frozen-semen programs are the one scenario where we tell every breeder, without exception, to use a quantitative reader. The 1.7-puppy gap from the 1,400-cycle study isn’t theoretical; it’s the cost of reading the number too coarsely.
FAQ
Which test confirms pregnancy — progesterone or relaxin?
Relaxin is the 1 pregnancy-only hormone: produced solely by the placenta, so it appears only in pregnancy and is reliably detectable from ~day 21–28, reaching 95–100% by day 30. Progesterone times ovulation but cannot confirm conception.
How early can I test for pregnancy?
Not before ~3 weeks — relaxin needs a placenta, and implantation hasn’t happened before roughly day 21. The earliest reliable detection is ~day 25 in some dogs, with near-total reliability by day 30.
Why do I need a number instead of a positive/negative strip?
Because the breeding decision lives in the number: 2–3 ng/mL is the LH surge, ~5 ng/mL is ovulation, 8–15 ng/mL is the window. A “positive” strip tells you the dog is cycling — which you already knew. And if the strip caps at 5 ng/mL, it can’t see the 10–20 ng/mL range frozen-semen timing requires.
How many tests does one breeding cycle actually take?
Five to eight serial progesterone tests, run every 1–2 days from early oestrus through ovulation, plus one or two relaxin tests around day 28–30 to confirm. Treat it as roughly one box of progesterone reagent per cycle.
What’s the difference between a semi-quantitative and a quantitative test?
Semi-quantitative brackets progesterone into coarse bands (often capped near 5 ng/mL); quantitative returns an exact concentration with pg/mL sensitivity and CV under 10%. For fresh-semen natural mating the coarse bands can work; for frozen or chilled semen you need the exact number.
Can progesterone confirm a pregnancy?
No — 1 timing marker, 0 pregnancy confirmation: progesterone rises in every oestrous cycle whether the bitch conceives or not; only relaxin (placental) confirms pregnancy.
What equipment do I need to run these tests?
1 analyzer, 2 channel options: the single-channel FIA680 suits breeders and small clinics; the six-channel FIA880 handles busy hospitals running multiple panels concurrently. Both read the full Migibio reagent menu.
Key Takeaways
- Two hormones, two jobs — progesterone times ovulation, relaxin confirms pregnancy. Confusing them is the #1 cause of wrong-test problems in breeding.
- “Positive” is not a result — every cycling bitch has progesterone; the decision lives in the number (2–3 ng/mL surge, ~5 ovulation, 8–15 window).
- Relaxin is the only pregnancy-only marker — reliably positive from ~day 21–28, 95–100% by day 30; before implantation there’s nothing to detect.
- Semi-quantitative caps at ~5 ng/mL — blind in the 10–20 ng/mL range that frozen semen (12-hour survival) demands.
- Precision pays — a 1,400-cycle study showed a 1.7-puppy gap (5.6 vs 3.9) between correctly-timed and mistimed frozen-semen breedings.
- Budget the program, not the kit — plan 5–8 progesterone tests plus 1–2 relaxin tests per cycle, roughly one reagent box.
References
- dvm360. “Breeding Management 102 — It’s All in the Timing (Proceedings).” Veterinary conference proceedings on progesterone thresholds and breeding-window timing.
- Kunanusont N, et al. “A comparison of progesterone determination methods in bitches.” 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 (ReproCHEK ELISA), as cited in PMC7126177.
- Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL). “Relaxin (Lateral Flow)” test guidance — detection windows and interpretation.
- PMC8924395. Comparative evaluation of three quantitative progesterone measurement methods in dogs.
This content is for educational and product-selection purposes only. Test results are one input to a breeding decision — always work with your veterinarian for diagnosis and insemination timing. Product specifications (cat. numbers, packaging, shelf life) are as published by Migibio (Guangzhou Magic Biotech Co., Ltd.) and may be updated; confirm with a sales representative before ordering.