1. EARLY PREGNANCY DETECTION IN DAIRY CATTLE BY BIOPRYN ™ ELISA TEST F. Toth, G. Gabor, Fan Huang and R.G. Sasser

2. INTRODUCTION PSPB is a novel protein that was first reported by scientists at the University of Idaho. PSPB is located in the giant binucleate cells of the trophoblastic ectoderm of the placenta and this indicated that it was either synthesized or sequestered by those cells. (Figure 1) Migration of these cells result the appearance of PSPB in the maternal circulation (Figure 2). PSPB is detectable in serum from 24 to 282 (parturition) days of gestation and can be applied reliably in dairy cow herds at 28 to 30 days after breading (Figure 3). The PSPB was found in sera of some primiparous cows as early as 15 days following conception and in sera of most cows at 24 days following conception.

3. The Giant Bi-Nucleated Cells Cotyledon Caruncle Figure 1:

4. Migration of the Giant Bi-Nucleated Cells Bi-Nucleated Cells Placenta Uterus PSPB in Circulation Figure 2:

5. PSPB ng/mL Parturition Days post breeding Variation of PSPB concentration in the serum Figure 3:

6. Complete clearence of the protein from blood may not occur until 80 to 100 days after parturition. For this reason testing of blood for a new pregnancy following parturition may give false positive tests. An ELISA test (BioPryn ™ by BioTracking LLC)has been developed for the examination of the PSPB in cattle 30 days after AI (at least 90 days after the previous parturition) (Figure 4 and 5). Decreasing the parturition interval is one of the most important issues in dairies. Pregnancy specific protein B (PSPB) is measured for detection of pregnancy in ruminants.

7. Figure 4: Biopryn ELISA test – color reaction

8. Figure 5: Biopryn ELISA test – stop color reaction

9. OBJECTIVES The objectives were: to verify the BioPRYN ™ test is adapted method for pregnancy detection in cattle. to determine the sensitivity of the BioPRYN ™ test and compare with the sensitivity of the ultrasonic examination. to define the practicability of PSPB test in Hungarian field. to exam the reliability of PSPB test in monitored circumstances.

10. METHODS Experiment 1 No. of cows No. of farms 69 1 The cows were examined by rectal ultrasonography between 29 and 55 days after AI. Blood samples were taken at the same time and were assayed for PSPB with BioPRYN ™ when all the samples has been collected.

11. Experiment 2 No. of cows No. of farms 1742 23 AI’s were carried out in 23 dairies and blood was drawn from 30 to 36 days after AI and assayed for early pregnancy by BioPRYN ™. To confirm the pregnancy diagnosis, rectal palpation was done on the farms at 45-90 days after AI.

12. Experiment 3 No. of cows No. of farms 336 1 In Experiment 3, blood samples were assayed with BioPRYN ™ for early pregnancy detection (30-36 days after AI) in 306 Holstein-Friesian and 30 Hungarian Fleckvieh cows. Confirmation was done by rectal ultrasonography one day after the blood collection.

13. RESULTS Distribution of Optical Density (OD) O D cutoff

14. Experiment 1

15. Experiment 2 Correct diagnosis: result of PSPB test corresponds with the result of ultrasonic examination. False diagnosis: result of PSPB test differs from the result of ultrasonic examination. Sensitivity: 100*correct positive diagnosis/correct positive diagnosis+false negative diagnosis. Specificity: 100*correct negative diagnosis/correct negative diagnosis+false positive diagnosis. Positive predicted value: 100*correct positive diagnosis/correct positive diagnosis+false positive diagnosis. Negative predicted value: 100*correct negative diagnosis/correct negative diagnosis+false negative diagnosis.

16. Experiment 3

17. DISCUSSION In addition, the more accurate ultrasound (and done more timely) rather than rectal palpation was used to confirm pregnancy. The rate of false positive tests was 5.9% and positive and negative prediction values were high (94.0 % and 100 %). The two highly controlled experiments (1 and 3) show that BioPRYN™ was an excellent test for pregnancy detection.

18. CONCLUSION In conclusion, the BioPRYN ™ test for PSPB is a practical and safe system for pregnancy detection in cattle.