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ubio quickVET RAPID Pregnancy Test for Cattle
Accurate diagnosis of pregnancy is of crucial importance in maintaining optimal reproductive performance of cattle and high profitability of dairy farms. It is desirable for the farmer to know as soon as possible if a mated or inseminated cow is not pregnant, so that she can be rebred with the minimum delay. It is most beneficial if non-‐pregnancy can be detected before the first heat after insemination. Conventional methods that allow early detection of pregnancy are not accurate enough, and reliance on these methods results in a large number of non-‐pregnant cows assumed to be pregnant and some pregnant cows being inseminated inappropriately, which could abort them. Conventional pregnancy detection methods that are accurate, on the other hand, can be performed only after at least 35 days after insemination, leading to the loss of at least one opportunity to inseminate.
ubio’s quickVET pregnancy detection test provides a perfect tool to detect non-‐pregnancy in cattle accurately within 21 days. The kit provides 100% detection of non-‐pregnant cases and allows insemination at the first opportunity. The average savings realised through the use of ubio quickVET test is as high as Rs.1300/-‐ per cow per lactation period. Moreover, the test can be easily performed within 20 minutes at cow-‐side without any instrumentation, which makes the tool attractive even for direct use by dairy farmers.
The test employs the well-‐tested progesterone assay principle to detect pregnancy. It measures progesterone levels in milk or serum (for heifers) using a Rapid ELISA technique.
COMPARISON OF PREGNANCY DETECTION TECHNIQUES
Test Stage Principle Advantages Disadvantages Accuracy Non-‐return to service
3 wks Recording dates of service and observation of symptoms of heat
-‐ Early detection, without missing first heat after insemination
-‐ Relies on good oestrus detection
-‐ Large variation within a population -‐ Insemination at ‘false oestrus’ can lead to abortion
About 50%
Early Conception Factor
4 wks + Detects pregnancy-‐dependent protein complex using ELISA
-‐ Relatively early detection
-‐ Needs blood -‐ May persist after loss of pregnancy
About 90%
Ultrasound Scan
4 wks + Visualization of images of the conceptus using realtime ultrasound
-‐ Relatively early detection -‐ Accurate
-‐ High capital cost -‐ Need good technical expertise
About 100%
Palpation 5-‐6 wks+
Feeling the presence of a fetus swelling in one of the uterine horns by inserting an arm into the rectum of the cow.
-‐ Accurate -‐ Possible damage to cow or conceptus -‐ Need good skill and technical expertise
About 100%
Oestrone Sulphate
15 wks+
Detects oestrone sulphate in milk. Concentration is proportional to oestrogens produced by conceptus.
-‐ Very accurate -‐ Too delayed to be useful in most cases
100%
Conventional 3 wks Progesterone -‐ Accurate -‐ High capital cost 100% for
ubio B i otec hnology Sys tems Pvt . Ltd. Ph : +91-484-2532966, +91-9446438678
Web: h t tp: //www.ubio.i n Emai l : c on tac [email protected] n
Milk Progesterone plate ELISA
concentration at 19-‐23 day interval from last heat is much higher in pregnant cows than non-‐pregnant ones
-‐ Early detection -‐ Non-‐invasive
-‐ Requires blood in heifers -‐ Test to be performed on specified days
non-‐pregnancy 85%+ for pregnancy
ubio quickVET Rapid Milk Progesterone Assay
3 wks Progesterone concentration at 19-‐23 day interval from last heat is much higher in pregnant cows than non-‐pregnant ones
-‐ Accurate -‐ Early detection -‐ Non-‐invasive -‐ No instrumentation -‐ Cow-‐side test
-‐ Requires blood in heifers -‐ Test to be performed on specified days
100% for non-‐pregnancy 85%+ for pregnancy
ASSAY PRINCIPLES
The ubio quickVET pregnancy test is based on ELISA-‐based progesterone assay principle. The test measures progesterone concentration in milk or blood using Rapid ELISA technique. A progesterone-‐specific monoclonal antibody is coated inside the test cassette. Milk/serum and labeled progesterone conjugate are added to the test and control wells during the test. The progesterone from the milk and the labeled conjugate will compete to bind with the coated antibody. A chromogen, that gives color, is added to the wells and it will bind only to the conjugate. If the sample contains progesterone (animal is pregnant), the test well will be lighter than the control well. If the progesterone level is very low (non-‐pregnant animal), the test well be bluer than the control well.
It is obvious that there is a significant difference between progesterone concentration in milk/blood samples taken from pregnant and non-‐pregnant cows from 19th to 23rd day after heat (and insemination). By checking the progesterone level during this time frame, we can accurately identify non-‐pregnancy.
This principle provides 100% accurate results for progesterone levels, which translates to 100% accuracy for non-‐pregnancy. However, there are some corner cases where a false positive may occur for cases marked ‘pregnant’ by the test.
1. If heat is incorrectly interpreted, and insemination is done at the wrong time, the test will show the result as ‘pregnant’ as we are actually detecting the high progesterone level during mid-‐heat, instead of that caused by pregnancy.
The concentration of progesterone in the milk or blood of a non-‐pregnant cow varies as shown below, with Day 0 corresponding to the occurrence of heat. It is assumed that insemination is appropriately performed at the time of heat.
In the case of a pregnant cow, the progesterone concentration varies as given below.
2. A cow that is correctly judged pregnant might lose her conceptus due to various reasons. In this case, the test is accurate, but the pregnancy does not result in calf-‐birth. It is suggested that the test be repeated if any sign of heat is seen after the cow is judged pregnant.
3. A cow with luteinized cystic follicles will produce progesterone at varying levels, and the test result is not reliable in such animals as it might show positive or negative based on the progesterone level on the day of test.
CLINICAL APPLICATIONS
1. Ovulation Test: ubio quickVET rapid pregnancy test for cattle can be used for detecting ovulation in cows that do not show regular symptoms of heat. The test should be performed during the suspected heat period. A dark blue spot indicates heat and insemination can be performed in 6-‐18 hours. A clear white card indicates mid-‐heat and the test needs to be repeated only after 10 days. A light blue color indicates approaching or past heat, and a repeated test after 2 days will confirm the condition.
2. Identification of follicular/luteal cysts: Ovarian cysts are an important infertility problem in dairy cattle. It is often difficult to differentiate ovarian cysts into follicular and luteal by palpation of the ovary per rectum. However, once the presence of an ovarian cyst has been determined by
palpation, differentiation can be made on the basis of progesterone concentration. Cystic cows having a low progesterone concentration are considered to have follicular cysts, and those with high concentrations are classified as having luteal cysts. For practical purposes, differential diagnosis of ovarian cysts is important in selection of endocrine therapy. The cyst can be differentiated by performing the test on two samples taken 7 days apart – a dark blue spot in both tests indicate a follicular cyst and a clear white card in both tests indicate a luteal cyst.
3. Evaluation of endocrine therapy: To evaluate treatment of follicular cysts using GnRH, ubio quickVET rapid pregnancy test can be used to evaluate to progesterone levels 10 days after administration of GnRH. A light blue spot or a clear white card indicates response to treatment. Similarly, the test can be used to evaluate response to treatment of luteal cysts using Prostaglandin compounds as unobserved estrus following prostaglandin administration is a common occurrence. Response to prostaglandin is dependent upon the presence of a functional corpus luteum. Therefore, milk progesterone testing prior to and 3 days after administration of prostaglandin can accurately assess if administration is justified (high concentration prior to administration) and if luteolysis has occurred (low concentration on day 3).
4. Evaluation of fitment for embryo transfer: ubio quickVET rapid pregnancy test for cattle can be used in embryo transfer programs to check if there is adequate progesterone level in the recipient to support the embryo. The recipient should show a light blue test result before an embryo is transferred.
INTERPRETATION OF THE RESULT
The control well is usually colorless or a very pale blue. A test well that is considerably bluer than the control well indicates a non-‐pregnant animal with low progesterone level. A test well lighter than or of the same shade as the control well indicates a pregnant animal with higher progesterone concentration, subject to caveats. Slightly bluer tinge in the test well when compared to the control well indicates non-‐pregnant result during approaching or past heat period – in this case, the test timing and heat calculation must be checked, and the test should be repeated if necessary.
Not Pregnant Not Pregnant Pregnant Pregnant
TEST PROCEDURE
1. Collect milk sample from cow in a clean vessel after discarding milk from the first few
pulls. For pregnancy test, the test should be done between 19th to 23rd day after insemination.
2. Keep the test pack at 26-‐30ºC for 1-‐2 hours to bring it to room temperature. Remove the test cassette from Aluminium pouch and keep
on a flat surface. There are two wells on the card, marked C and T.
3. Add three (3) drops of milk to both the wells
(C and T) using a dropper supplied with the test. Dispose the dropper immediately.
4. Keep the cassette on a flat surface for 5
minutes. Periodically tap the side of the cassette for proper mixing. Take care not to splash the milk in the wells.
5. Using a fresh dropper, Add three (3) drops of reagent into both the wells (C and T) from the bottle marked Reagent 1. Let milk and
reagent mix by gently tapping the cassette.
6. Keep the cassette on a flat surface for 20 minutes. Gently tap the cassette at 4-‐minute
intervals.
7. Tip the cassette and empty the wells into a sink.
8. Fill the wells with the wash buffer in the dropper bottle marked ‘Reagent 2’, tap gently and empty the wells into the sink. Repeat this
washing 5-‐6 times. Shake off any remaining solution from the wells.
9. Add 5 drops of Reagent 3 in the opaque vial
to both the empty wells. Keep the test on a flat surface. The result appears after 8 minutes, and can be read till 15 minutes.
KIT COMPONENTS -‐ One Aluminium pouch containing the test cassette.
-‐ Three droppers for transferring milk and reagents.
-‐ One transparent vial marked ‘1’
containing reagent 1.
-‐ One dropper bottle marked ‘2’ containing wash buffer (reagent
2).
-‐ One opaque vial marked ‘3’ containing reagent 3
PRECAUTIONS & WARNINGS -‐ Store in fridge at 4-‐8ºC, do not freeze
-‐ Do not reuse test kit.
-‐ Use a separate dropper for each reagent.
-‐ Do not mix components from
different kits.
-‐ Do not touch the test wells.
