Frequently Asked Questions
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​PAG is shorthand for Pregnancy-Associated Glycoprotein.
The PAG test which has been developed by IDEXX is called the Alertys Pregnancy Tests*. It is an enzyme-linked immunosorbent assay (ELISA) test. It has been developed to accurately detect PAG molecules in blood, serum or milk. It is validated to be used for pregnancy diagnosis in cattle, sheep, goats, water buffalo and bison. Unlike other chemical tests for pregnancy using progesterone as an indicator, the PAG test is very specific for pregnancy.
*The Alertys Rapid Visual and OnFarm pregnancy tests can be run without ELISAÂ instrumentation.Â
PAGs are detectable as early as 28 days after insemination, so it is recommended that the test can be used from this point and throughout the whole gestation period.
Progesterone is a hormone that occurs with peaks and valleys during the normal reproductive cycle and is not pregnancy specific.
The Alertys pregnancy tests detect pregnancy-specific proteins called pregnancy-associated glycoproteins (PAGs). These specific proteins are expressed in the embryonic regions of the placenta and detected in maternal fluids such as blood and milk.
The Alertys Milk Pregnancy Test uses milk samples collected during routine milk recording or collected from self-sampling pots, which will be most suitable for dairy farms. The test cannot be performed on-farm as specialist laboratory equipment is required.
The Alerty Ruminant Pregnancy Test, which uses blood samples, on the other side, may be more convenient for beef herds. Again, the samples are tested in a lab as specialist equipment is required.
For smaller-scale testing organisations, such as individual veterinary practices, the Alerty Rapid Visual Pregnancy Test can be used. A blood or serum sample is required, and the test can be completed in around 30 minutes. Some technical skill is required, and this isn’t a cow-side test, but there is no necessity for specialised laboratory equipment to observe the colour change to make a pregnancy diagnosis.
Finally, there is an Alertys OnFarm Pregnancy Test, which was recently introduced in 2021. This lateral-flow style test can be used on farm and requires no technical expertise, other than to collect a blood sample (EDTA sample). A measured amount of blood is dropped onto the test cartridge, and a pregnancy diagnosis result is ready in a matter of minutes, not unlike a human home-pregnancy test kit. This is suitable as a cow-side test.
Open cows can have a major impact on profitability.
Research shows that the average cost per open dairy cow is €5 a day*. These costs include the value of the milk the cow would have produced, the value of her calf, and other relevant factors. Dairy farms can optimise profitability by shortening the calving interval and reducing the number of days that cows are open.
For beef farmers, shorten calving intervals with safe, ongoing confirmation of pregnancy status at critical stages, saves an estimated $77 per head** by identifying and removing sub-fertile cows early.
* De Vries et al, Economics of improved reproductive performance in dairy cattle (Publication AN 156), Gainesville FL, University of Florida Institute of Food and Agriculture Science; 2005
** Cook B, Biermacker JT, Childs D. The value of pregnancy testing beef cows. Paper presented at: 2007 Southern Agricultural Economics Association Annual Meeting; February 4–7, 2007; Mobile, Alabama.
During the normal gestation process of a cow, approximately 10-25% of pregnancies will be lost between conception and full-term due to unknown causes. Pregnancy testing at specific times throughout gestation helps improve reproductive efficiency by finding cows who have experienced pregnancy loss.
It is recommended to test 2-3 times throughout gestation; first at 28-30 days post- breeding, again at 60 days post-breeding, and before dry off to be sure only pregnant cows are kept on farm.
It is clear there is a strong case for early and accurate pregnancy diagnosis in dairy herds. The method used for pregnancy diagnosis is somewhat a matter of personal choice, depending on individual circumstances and convenience.
Both transrectal ultrasound (TRUS) and PAG testing are reliable methods of early pregnancy diagnosis. Most dairy farms find that working closely with a progressive veterinary practice carries many benefits. This remains the case whether you choose to use the Vet for TRUS pregnancy diagnosis, or not. Of course, transrectal ultrasound can play a role in other areas of fertility management too, such as diagnosis of non-cycling or cystic cows. A mix-and-match approach can be a sensible way forwards for many herds, taking advantage of PAG pregnancy diagnosis for its timeliness, simplicity, and accuracy, whilst reducing the numbers of cows needing to be separated and presented at the routine vet visits. Some farms find this then frees up their Vet to focus on valuable tasks, such as fertility data analysis, training, and other beneficial work.
Yes, PAGs will circulate in milk and blood for a certain period of time after embryonic loss or abortion. For early embryonic loss, we estimate PAGs will disappear within 6-10 days. In case of late term abortion PAGs may be present for a longer period of time (40-60 days). It should be noted that it can take up to 60 days for PAGs to drop below the test threshold post-calving.
You can still use one of the Alertys tests, you just need to apply some rules around testing. For example, test 50 days after the cow is put with bull (21 days for heat plus 28 days to earliest test), or test 28 days after the bull is removed from the herd.
If the bull is running with the herd all the time, you may need to test monthly, BUT remember, the test will not tell you how far in calf the cow is. And a negative result does not mean not-pregnant if test is done less than 28 days since service.
PAG Testing
The Alertys Milk Pregnancy Test has been developed to perform robustly on milk samples from cows, goats, sheep and water buffalo. The test can be run on samples that have been exposed to heat treatment (during milk component analysis) and preserved milk samples (e.g. Bronopol). For more information on the types of preservatives that have been validated with this test, please contact your IDEXX technical services representative. The assay can be run on whole or skim milk samples. Samples should not be micro-waved prior to testing.
PAGs are detectable as early as 28 days after insemination.
PAG levels take time to disappear after calving or embryonic loss. We recommend leaving 60 days after calving and up to 10 days after early embryonic loss before using the test. The further on in gestation then the longer it takes for the PAG level to drop below the cut off line.
Yes, if certain conditions are met.
Excellent results obtained on well-parameterised Lely* robot:
Ensure sampling shuttle is installed correctly horizontally (verify with a spirit level). Please also ensure that the length of hose is well adjusted.
Increase emptying time of the milk pump ≥ 9 seconds (do not forget to reset the parameters to the initial setting after the weighing).
The results of reliability are very similar to the values obtained from a normal milking parlour (only 3.5% of samples to be re-checked and 2% false positives).
*Test in progress on other brands
Whatever the method of pregnancy diagnosis, it is useful to do a confirmatory test after around 70-110 days post breeding*. The earlier the initial pregnancy test, the more important the confirmatory test is.
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The value of doing an initial pregnancy test early is to quickly identify the open cows so they can be re-bred. Most open cows should have been detected in oestrus at the 3-week return point. However, some balance needs to be struck. If we test too soon, a greater proportion of pregnancies will be lost (a confirmatory test is definitely required) and if we test too late, we aren’t getting the advantage of finding those open cows to serve again.
*LeBlanc, S. J. 2013. Short communication: Field evaluation of a pregnancy confirmation test using milk samples in dairy cows. J. Dairy Sci. 96:2345-2348.
Poor quality samples may compromise the accuracy of test results. Bronopol or a similar preservative may be used to maintain sample quality. Prior to testing, milk samples should be checked to ensure that they are not soured or separated and are free from contamination. Care should be taken to minimise the likelihood of milk carry-over from cow to cow during sample collection, particularly when using samples collected for routine herd recording
There is some evidence to suggest that higher milk yields result in lower circulating PAG levels, but this does not affect test results. There is currently no evidence to suggest that other milk components have an effect on test results. Also, IDEXX has no evidence to date to suggest that antibiotic treated milk has a negative effect on the test results.
Early embryonic loss can occur in 10-20% of previously confirmed pregnancies. You can use the milk pregnancy test to confirm pregnancy (to detect early embryonic losses) and to prevent dry-off of open cows. There is no extra hassle and in many instances your milk laboratory can automate this testing for you. So, you are alerted if a cow needs attention and you can have the Vet out to check. Many Milk Recording Organisations/Labs also offer milk pregnancy testing anytime and have a sample kit available for you to collect your own samples.
Yes, frozen and thawed samples can be run on the Alertys Milk Pregnancy Test. Make sure to thoroughly mix the thawed sample. Mix by gentle vortexing or inverting at least five times. Frothing or overmixing of samples will cause denaturation of proteins. If a sample is frozen and thawed repeatedly, the sample quality may suffer due to clotting, and test results may be altered.
Alertys Milk Pregnancy Test
Serum samples with trace hemolysis (light-red color) and moderate lipemia (milky appearance) may have little or no effect on ELISA results.
Avoid using samples that are heavily hemolyzed (dark-red color) or grossly lipemic. When serum is on the clot, be careful not to aspirate any of the clotted material or blood cells.
The Alertys Ruminant Pregnancy Test delivers reliable blood-based results that support early, confident decisions. Shorten calving intervals with safe, ongoing confirmation of pregnancy status at critical stages. Save an estimated $77 per head by identifying and removing subfertile cows early*
*Cook B, Biermacker JT, Childs D. The value of pregnancy testing beef cows. Paper presented at: 2007 Southern Agricultural Economics Association Annual Meeting; February 4–7, 2007; Mobile, Alabama.
Pregnancy check 1 (P1): 28−35 days of gestation, after artificial insemination (AI); the first opportunity to find non-pregnant (open) cows. Estrous synchronisation programs allow these cows to be rebred more quickly.
Pregnancy check 2 (P2): 35−70 days of gestation; peak period of early embryonic death (EED). Best-practice management programs confirm pregnancy status and identify EED.
Pregnancy check 3 (P3): 90−110 days of gestation; EED peak is now past. The cow is approaching the break-even phase of the lactation curve. Testing can help you make breeding/culling decisions.
Pregnancy check 4 (P4): 200−230 days of gestation (dry-off). Although uncommon, pregnancy loss can occur between 100−230 days of gestation. This is an important decision point for dry-off and culling.
Yes, frozen and thawed serum samples can be run on the Alertys Ruminant Pregnancy Test. But only serum samples should be frozen, not whole blood. Make sure to thoroughly mix the thawed sample. Mix by gentle vortexing or inverting at least five times. Frothing or overmixing of samples will cause denaturation of proteins. If a sample is frozen and thawed repeatedly, the sample quality may suffer due to clotting, and test results may be altered.
Alertys Ruminant Pregnancy Test
Alertys OnFarm Pregnancy Test
The Alertys Rapid Visual Pregnancy Test is a bovine whole blood (EDTA), plasma (EDTA) or serum pregnancy test, which detects pregnancy associated glycoproteins (PAGs).
The kit holds up to 25 samples at a time and delivers results in 21 minutes assay time. It can used on-farm or in-clinic without needing ELISA equipment to run.
The test detects open cows from 28 days of pregnancy and throughout gestation. It allows veterinarians and producers to quickly re-breeding open cows and improve reproductive efficiency.
Both kits determine pregnancy status in bovine serum/plasma from 28 days post-breeding.
We recommend using the Alertys Ruminant Pregnancy Test if you already have ELISA instruments or if you plan to test more than 10,000 samples each year.
If you do not have ELISA instruments or plan to test fewer than 10,000 samples, we recommend the Alertys Rapid Visual Pregnancy Test.
All reagents required to run the test are provided in the kit. Two 96 well plates with 1x8 strips, a negative control, a positive control, 4 reagents, dropper caps, pipette tips and a use instruction.
You might also want to order an accessory pack when you order the Rapid Visual Pregnancy Test the first time. This contains a wash bottle, a plate cover and a precision pipette.
The only thing you need to buy in addition is distilled or deionized water to be used for the wash steps.
Contact your IDEXX representative or fill out this form to order the kit or request more information.
The product code of the Alertys Rapid Visual Pregnancy Test is 99-41369. The product code of the accessory pack is 99-0005524.
The Alertys Rapid Visual Pregnancy Test detects pregnancies when embryos are 28 days old. In the case of embryo transfer (ET), this is 21 days after ET assuming embryos were transferred at 7 days of age.
IDEXX has data on file that shows that the Alertys Ruminant Pregnancy Test detects pregnancies 21 days post ET. IDEXX is currently initiating studies to show detection of pregnancies 21 days post ET with the Alertys Rapid Visual Pregnancy Test.