1. USE OF PREGNANCY SPECIFIC PROTEIN B TEST FOR PREGNANCY DIAGNOSIS AND ASSESMENT OF BULL FERTILITY G. Gábor, F. Tóth, N. Solymosi

3. 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. Cotyledon Caruncle

4. Migration of these cells result the appearance of PSPB in the maternal circulation. Bi-Nucleated Cells Placenta Uterus PSPB in Circulation

5. PSPB ng/mL Parturition Days post breeding 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.

6. 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. 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).

7. Biopryn ELISA test – color reaction

8. Biopryn ELISA test – stop color reaction

9. OBJECTIVES Decreasing of the parturition interval and checking bull fertility are both important issues in dairies. Measuring of Pregnancy Specific Protein B (early pregnancy detection) appears to be a useful tool assess the true fertility of breeding bulls. 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 and in monitored circumstances. to exam the reliability of PSPB test for the bull fertility. checking the prediction value of PSPB test for the embryonic loss.

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 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.

12. Ultrasonic appearance of the bovine fetus at different stages of the pregnancy

13. Experiment 3 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 60 days after AI.

14. Experiment 4 No. of bulls No. of farms 8 3 No. of AI: 816 Semen was collected from the 8 bulls and (61-139 inseminations per bull) AI’s were carried out in 3 dairies. 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 60 days after AI.

15. RECENT EXPERIMENTS Routinely collected blood sample from 3 farms for almost 1 year, and checking pregnancy status by PSPB test 30-36 days after AI. Open cows and cows with low OD of PSPB (range: ±10 % of cutoff) were assayed for serum P4. Confirmation of the pregnancy was done by rectal palpation and the most important reproduction, production and breeding information were also collected in a data base.

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

17. Experiment 1 100 % agreement was found between the serum PSPB and ultrasonic examinations

18. 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.

19. Experiment 3

20. Experiment 4 BullPregnantOpenTotalEmb. loss % Z859949.6 G73108312.0 S1111812914.0 F1192013914.4 W991811715.4 T61127316.4 V50116118.0 R972312019.2 TOTAL:69512181614.8Mean

21. Prediction of the embryonic loss by Biopryn and serum P4 (I. total loss) Number of pregnancies2081 Predicted embryonic loss206 True embryonic loss10149.03% Non predicted loss23912.75% Total embryonic loss34016.33%

22. Prediction of the embryonic loss by Biopryn and serum P4 (II. Based on serum P4 level) Serum P4 level groups True embryonic loss False embryonic loss High P4 serum level (>4 ng/ml)2750 Medium P4 serum level (2-4 ng/ml)4142 Low P4 serum level (<2 ng/ml)3313

23. Prediction of the embryonic loss by Biopryn and serum P4 (III. based on milk production) Group (kg milk) No. cows No. calving Milk (kg) Age (year) Day after calving Embr. loss% 0-202322.415.54.3211.812.93 20-307352.125.43.8157.514.69 30-408532.334.23.9125.517.47 40-502482.744.14.3100.820.56

24. Prediction of the embryonic loss by Biopryn and serum P4 (IV. Based on loss of AI bull groups) Category (loss of AI bulls) No. bullsPregnanciesPregnantOpen 0-10 % (mean: 2.9 %)61721675 10-20 % (mean: 14.7 %)15712607105 >20 % (23.3 %)11545418127 Total3214291192237

25. Prediction of the embryonic loss by Biopryn and serum P4 (V. based on the loss of offspring) Group (loss%) father bulls0-10 %10-20 %> 20 %Total Bulls27 3185 Examined3744975091380 Pregnant cows13.115.712.7 Open cows0.82.74.3 Non predicted loss0.41.83.0 Predicted loss1.11.82.1 True loss0.40.91.3 Parturition /cow1.92.22.4 AI /cow2.72.52.7 Day after calving148.5135.7142.5 Milk kg/cow28.930.831.2 Age (year)3.53.94.1 Embryonic loss %4.114.226.5

26. The two highly controlled experiments (1 and 3) show that BioPRYN™ was an excellent test for pregnancy detection, that’s why BioPRYN ™ test for PSPB is a practical and safe system for pregnancy detection in cattle. It looks also useful to check the real bull fertility. It may have a role (combining with the control of the serum P4 level) in prediction of the embryonic loss as well.