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

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

INTRODUCTIONPSPB 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.

The Giant Bi-Nucleated CellsCotyledonCaruncleFigure 1:

Migration of the Giant Bi-Nucleated CellsBi-Nucleated CellsPlacentaUterusPSPB inCirculationFigure 2:

PSPB ng/mLParturitionDays post breedingVariation of PSPB concentration in the serum Figure 3:

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.

Figure 4: Biopryn ELISA test color reaction

Figure 5: Biopryn ELISA test stop color reaction

OBJECTIVESThe 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.

METHODSExperiment 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.

Experiment 2No. of cows No. of farms 1742 23

AIs 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.

Experiment 3No. 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.

RESULTS

Distribution of Optical Density (OD) O Dcutoff

Chart1

0.548

0.501

0.486

0.479

0.458

0.457

0.448

0.427

0.422

0.416

0.41

0.405

0.402

0.4

0.392

0.39

0.382

0.373

0.369

0.362

0.358

0.357

0.347

0.341

0.336

0.334

0.321

0.309

0.303

0.302

0.295

0.288

0.276

0.265

0.262

0.258

0.235

0.227

0.2190282376

0.194

0.19

0.144

0.12

0.114

0.114

0.108

0.106

0.102

0.102

0.1

0.1

0.099

0.099

0.096

0.093

0.091

0.089

0.087

0.087

0.086

0.086

0.085

0.084

0.084

0.083

0.082

0.082

0.082

0.081

0.08

0.078

0.077

0.077

0.072

Sheet1

Sheet1

0.548

0.501

0.486

0.479

0.458

0.457

0.448

0.427

0.422

0.416

0.41

0.405

0.402

0.4

0.392

0.39

0.382

0.373

0.369

0.362

0.358

0.357

0.347

0.341

0.336

0.334

0.321

0.309

0.303

0.302

0.295

0.288

0.276

0.265

0.262

0.258

0.235

0.227

0.2190282376

0.194

0.19

0.144

0.12

0.114

0.114

0.108

0.106

0.102

0.102

0.1

0.1

0.099

0.099

0.096

0.093

0.091

0.089

0.087

0.087

0.086

0.086

0.085

0.084

0.084

0.083

0.082

0.082

0.082

0.081

0.08

0.078

0.077

0.077

0.072

Sheet2

Sheet3

Experiment 1

Munka1

1. Tbl.: Az ELISA teszt s az ultrahangos vemhessgvizsglat

eredmnyeinek sszevetse

Az ELISA teszt elzetesen meghatrozott hatrrtke: 0,325

vizsglati naptermkenytsvemhessgi id (nap)UH diagnzisELISA teszt eredmnyeELISA diagnzis

5/15/014/14/0131vemhes1.024vemhes

5/15/014/9/0136res0.225res

5/15/014/10/0135res0.224res

5/15/014/9/0136vemhes0.783vemhes

5/15/014/12/0133res0.242res

5/15/014/11/0134vemhes0.965vemhes

5/15/014/13/0132vemhes0.945vemhes

5/15/014/10/0135res0.227res

5/15/014/14/0131res0.261res

5/15/014/16/0129vemhes0.908vemhes

5/15/014/7/0138res0.199res

5/15/014/6/0139vemhes0.727vemhes

5/15/014/15/0130vemhes0.661vemhes

5/15/013/21/0155res0.192res

5/15/014/11/0134vemhes0.862vemhes

5/23/014/11/0142vemhes1.109vemhes

5/23/014/21/0132res0.179res

5/23/014/22/0131res0.234res

5/23/014/21/0132res0.204res

5/23/014/15/0138vemhes0.704vemhes

5/23/014/14/0139res0.189res

5/23/014/20/0133vemhes0.834vemhes

6/6/015/4/0133res0.278res

6/6/015/1/0136vemhes0.992vemhes

6/6/015/2/0135res0.198res

6/6/014/19/0148vemhes1.023vemhes

6/6/015/4/0133res0.183res

6/6/015/3/0134vemhes0.717vemhes

6/6/015/3/0134vemhes0.709vemhes

6/6/015/4/0133vemhes0.901vemhes

6/6/015/3/0134res0.23res

6/6/015/1/0136res0.193res

6/6/014/13/0154vemhes0.924vemhes

6/6/015/4/0133vemhes0.66vemhes

6/6/015/4/0133vemhes0.888vemhes

6/6/015/4/0133res0.19res

6/6/015/4/0133vemhes0.626vemhes

6/6/015/3/0134res0.168res

6/6/014/15/0152res0.177res

6/6/015/1/0136res0.192res

6/6/015/2/0135vemhes1.154vemhes

6/6/015/3/0134vemhes0.81vemhes

6/12/015/2/0141res0.262res

6/12/015/6/0137vemhes1.169vemhes

6/12/015/1/0142res0.177res

6/12/015/10/0133res0.176res

6/12/015/3/0140res0.164res

6/12/015/3/0140res0.171res

6/12/015/5/0138vemhes0.872vemhes

6/12/015/9/0134res0.21res

6/12/015/3/0140vemhes0.894vemhes

6/12/015/10/0133res0.286res

6/12/015/3/0140res0.219res

6/12/015/2/0141vemhes0.706vemhes

6/12/015/3/0140res0.207res

6/12/015/10/0133vemhes1.04vemhes

6/12/015/10/0133res0.178res

6/12/015/6/0137res0.206res

6/12/015/5/0138res0.198res

6/12/015/9/0134vemhes1.247vemhes

6/12/015/10/0133vemhes1.004vemhes

6/12/015/9/0134res0.254res

6/12/015/9/0134vemhes1.405vemhes

6/12/015/8/0135res0.175res

6/12/015/3/0140vemhes1.124vemhes

6/12/015/7/0136res0.197res

6/12/015/6/0137res0.211res

6/12/015/10/0133res0.308res

6/12/015/6/0137vemhes0.955vemhes

Munka2

vemhessgi id (nap)ultrahang diagnzisELISA teszt eredmnyeELISA diagnzis

31res0.261res31res0.261

31res0.234res31res0.234

32res0.179res32res0.179

32res0.204res32res0.204

33res0.242res33res0.242

33res0.278res33res0.278

33res0.183res33res0.183

33res0.19res33res0.19

33res0.176res33res0.176

33res0.286res33res0.286

33res0.178res33res0.178

33res0.308res33res0.308


34res0.168res34res0.168


34res0.254res34res0.254

35res0.224res35res0.224

35res0.227res35res0.227

35res0.198res35res0.198

35res0.175res35res0.175

36res0.225res0.22025

36res0.193res0.168

36res0.192res0.308

36res0.197res

37res0.206res36res0.225res













29vemhes0.908vemhes41res0.262res




33vemhes0.834vemhes0.1987647059

33vemhes0.901vemhes0.164


33vemhes0.888vemhes

33vemhes0.626vemhes29vemhes0.908



















40vemhes1.124vemhes0.9136842105



48vemhes1.023vemhes


36vemhes0.992

37vemhes1.169

37vemhes0.955

38vemhes0.704

38vemhes0.872

39vemhes0.727

31res0.26140vemhes0.894

31res0.23440vemhes1.124

32res0.17941vemhes0.706

32res0.20442vemhes1.109

33res0.24248vemhes1.023

33res0.27854vemhes0.924

33res0.1830.9216923077

33res0.190.704

33res0.1761.169

33res0.286

33res0.178

33res0.30829vemhes0.908vemhes


























0.2140vemhes0.894vemhes



42vemhes1.109vemhes

48vemhes1.023vemhes

54vemhes0.924vemhes

0.92

0.63

1.41

Munka3

OD values

MeanMinimumMaximum

Days After AIopenpregnantopenpregnantopenpregnant

29 - 350.220.910.170.6260.311.41

n= 20n= 19

36 - 550.200.920.160.7040.261.17

n= 17n= 13

Total0.210.920.160.630.311.41

n= 37n= 32

Cutoff:0.325

Experiment 2Correct 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.

Sheet1

Number of AI1742

Correct positive diagnosis772

Correct negative diagnosis638

False positive diagnosis292

False negative diagnosis40

Sensitivity95.1

Specificity68.6

Positive predicted value72.6

Negative predicted value94.1

Sheet2

Sheet3

Experiment 3

Sheet1

Number of AI1742



Fals positive diagnosis292

Fals negative diagnosis40

Sensitivity95.1

Specificity68.6


Negative predicted value94.1

Number of AI336



Fals positive diagnosis20

Fals negative diagnosis0

Sensitivity100

Specificity89.1


Negative predicted value100

Sheet2

Sheet3

MBD00152438.xls

Sheet1

termkenyts szma1742

korrekt pozitv772

korrekt negatv638

fals pozitv292

fals negatv40

rzkenysg95.07

negatv elrejelzs94.1

Termkenyts336

Korrekt pozitv316

Korrekt negatv164

Fals pozitv20

Fals negatv0

rzkenysg100

Pozitv elrejelzs94

Negatv elrejelzs100

Sheet2

EmbryonicEmbryonicTotalTotalEmbryonic loss

Pregnantlosslossno. of losslossbetweenMilk kg /Correlation

FarmsNo. of cowsbetweenbetweenbetween%25-36 days36-60 dayscow/year

25-36 days36-60 days25-60 days%

I.1908212915.34.211.18000milk / total loss0.73

II.2515444919.5217.59500milk / loss (25-36 days)-0.36

III.175215179.71.18.67500milk/ implantation0.91

IV.1617172414.94.310.67000

Total777229711914.82.911.98000

DISCUSSIONIn 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.

CONCLUSION

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

Documents

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