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Slide 1: Pregnancy Diagnosis in Cow © 2008 ubio. All rights reserved. 1
Slide 2: Introduction Accurate diagnosis of pregnancy is of crucial importance in maintaining optimal reproductive performance of cattle and high profitability of dairy farms. 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 © 2008 ubio. All rights reserved. 2
Slide 3: ubio’s quickVET pregnancy detection test  Detect non-pregnancy in cattle accurately within 21 days. 100% detection of non-pregnant cases and allows insemination at the first opportunity.  The average savings realized through the use of ubio quickVET test is as high as Rs.1300/- per cow per lactation period.   The test can be easily performed within 20 minutes Cow-side test without any instrumentation, which makes the tool attractive even for direct use by dairy farmers.  It measures progesterone levels in milk or serum (for heifers) using a Rapid ELISA technique.  © 2008 ubio. All rights reserved. 3
Slide 4: COMPARISON OF PREGNANCY DETECTION TECHNIQUES Te s t N o n-re turn to s e rv ic e S ta g e 3 wk s P rinc ip le Re c o rd ing date s o f s e rv ic e and o b s e rvatio n o f s ym pto m s o f he a t A d va n ta g e s - Early de te c tio n, wit ho ut m is s ing f irs t he at afte r ins e m inatio n D is a d va nt a g e s - Re lie s o n go o d o e s trus de te c tio n - Large v ariat io n wit h in a po pu latio n - Ins e m inatio n at ‘fals e o e s trus ’ c a n le a d to ab o rtio n © 2008 ubio. All rights reserved. 4
Slide 5: 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.  If the sample contains progesterone (animal is pregnant), the test card will be completely white. If the progesterone level is very low (non-pregnant animal), a bright blue colour will appear on the card.  © 2008 ubio. All rights reserved. 5
Slide 6: • The concentration of progesterone in the milk or blood of a nonpregnant cow varies as shown. 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 shown. © 2008 ubio. All rights reserved. 6
Slide 7: Test Procedure © 2008 ubio. All rights reserved. 7
Slide 8: 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  © 2008 ubio. All rights reserved. 8
Slide 9: 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..  © 2008 ubio. All rights reserved. 9
Slide 10: Keep the test pack at 2630ºC for 1-2 hours to bring it to room temperature.  Remove the test cassette from pouch and keep on a flat surface.  There are two wells on the card, marked C and T.  © 2008 ubio. All rights reserved. 10
Slide 11: Add three (3) drops of milk to the test well (marked T) using a dropper supplied with the test.   Dispose the dropper immediately. Add one (1) drop of Reagent 2 to the control well (marked C).  © 2008 ubio. All rights reserved. 11
Slide 12: • Using a fresh dropper, Add three (3) drops into both the wells (C and T) from the bottle marked Reagent 1. Let milk and reagent mix by gently tapping the cassette. Take care not to splash the liquid in the wells. • Keep the cassette on a flat surface for 20 minutes. Gently tap the cassette at 4-minute intervals. © 2008 ubio. All rights reserved. 12
Slide 13: • Tip the cassette and empty the wells into a sink. • Fill both the wells (C and T) with the wash buffer in the 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. © 2008 ubio. All rights reserved. 13
Slide 14: • Add 5 drops of Reagent 3 in the opaque vial to both the empty wells (C and T). • Keep the test on a flat surface. • The result appears in 10 minutes, and can be read till 15 minutes. • DO NOT READ THE RESULT AFTER 15 MINUTES. © 2008 ubio. All rights reserved. 14
Slide 15: Result © 2008 ubio. All rights reserved. 15
Slide 16: Not Pregnant Test well (T) bluer than control well (C) Pregnant Test well (T) same as or lighter than control well (C) © 2008 ubio. All rights reserved. 16
Slide 17: • This principle provides 100% accurate results for progesterone levels, which translates to 100% accuracy for non-pregnancy. Test Non­return to service Stage 3 wks Principle Recording dates of service and observation of symptoms of heat  Advantages ­ Early detection, without missing first heat after insemination Disadvantages ­ Relies on good oestrus detection ­ Large variation within a population Accuracy About 50% However, there are some corner cases where a­ Insemination at false positive may occur for cases marked ‘pregnant’ by the test. They are............................. © 2008 ubio. All rights reserved. 17
Slide 18: 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. 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 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. © 2008 ubio. All rights reserved. 18
Slide 19: Clinical aplications 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 bright blue well indicates heat and insemination can be performed in 6-18 hours. A clear white well 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. © 2008 ubio. All rights reserved. 19
Slide 20:  Identification of follicular/luteal cysts:  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 bright blue well in both tests indicate a follicular cyst and a clear white card in both tests indicate a luteal cyst. Follicular Cyst Luteal Cyst  Day 1 Day 7 © 2008 ubio. All rights reserved. Day 1 Day 7 20
Slide 21: To evaluate treatment of follicular cysts using GnRH,  Evaluate response to treatment of luteal cysts using Prostaglandin  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. 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). Day 0 © 2008 ubio. All rights reserved. Day 3 21
Slide 22: •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. © 2008 ubio. All rights reserved. 22
Slide 23: For further details and placing orders ubio Biotechnology Systems Pvt Ltd. XII/111-E/F, Biotechnology Incubation Center Kinfra Hi-Tech Park, Kalamassery Cochin, India 683503 Ph: +91-484-2532966, +91-9446438678, +91-9744122269 Email: contact@ubio.in www.ubio.in © 2008 ubio. All rights reserved. 23
Slide 24: www.ubio.in contact@ubio.in © 2008 ubio. All rights reserved. 24