Diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in the plasma of dairy cows

TMERIOGENOLOGY

DIAGNOSIS OF PREGNANCY BY RADIOIMMUNOASSAY OF A PREGNANCY-SPECIFIC PROTEIN IN THE PLASMA OF DAIRY COWS P.Humblot,'S.Camous,'J. Martal,2J.Charlery,1 N.Jeanguyot,' M.Thibierland G.Sasser3 IUNCEIA, Laboratoire d'Hormonolcqie, 13 Rue Jouet, B.P.65 94703 Maisons Alfort, France 2INRA, CNRZ Unite d'Endocrinologie de l'embryon 78350 Jouy en Josas, France 3, University of Idaho Department of Animal Science Moscow, Idaho 83843 USA Received for publication: April 15, 1987 Accepted: Mug 18, 1988 ABSTRACT The accuracy and efficiency of progesterone (P4 ) and bovine pregnancy-specific protein B (bPSPB) radioimmunoassays (RIA) in detectirq pregnant and nonpregnant dairy cows were compared at different stages of pregnancy. The study included 145 French Friesian heifers and cows from a single herd. A total of 175 artificial insemination (A.1.) and blood sampling procedures were performed. Animals were bled 24 d post AI for P4 RIA. They were bled at 24,26,30 to 35, and 70 f 9 after AI for bPSPB RIA. Females were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB RIA, cows were nonpregnant when at least one of the B/Be x 100 replicates was higher than 95% in the RIA. When compared with palpations per rectum at 70 d, the accuracy of positive diagnoses (no. positive and pregnant/no. positive diagnoses) by P4 RIA at Day 24 was 67.2% (82/122). The accuracy of negative diagnoses was 98% (52/53). Accuracy of positive diagnoses by bPS?B RIA increased with gestation age (P
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INTRODUCTION Early detection of pregnancy by specific placental and/or embryonic glycoproteins has been performed for many years in the human using hCG assay (1). In farm animals, similar specific pregnancy markers involved in the maintenance of the corpus luteum (CL) have been found (2,3), but none of them was assayable in peripheral plasma or milk. In these species, and mare specifically in the cow, early pregnancy diagnosis is based on the use of P4 RIA (4-Y) or enzyme immunoassay (EIA) rn milk or plasma (10-14). Recently, bPSPB, secreted by binucleate trophoblastic cells (15), was isolated from placental membranes (16). Development of an RIA has allowed the determination of bPSPB concentration in cow serum throughout pregnancy (17). First, experiments were conducted in beef cattle to test the accuracy of the bPSPB assay as a method for pregnancy diagnosis (17). Results were very satisfactory: 99 and 95%, respectively, of the negative and positive results were accurate, but the period of gestation, when the test can first be used, was not determined because breeding and conception dates were not known. Cur study was conducted on dairy cattle to compare the accuracy of the bPSPB RIA when performed at specific intervals after AI with that obtained from plasma P4 RIA at Day 24.

MATERIALS AND METHODS Animals and Treatments Seventy-six cows and 69 heifers (18 to 24 mo old) of the French Friesan breed were kept in free stall areas. Cows had 1 to 8 lactations; average milk production of multiparous cows (n = 58), estimated from the data of the previous lactation, was 5141 + 163 kg milk (Z f SEM). Cows were artificially inseminated at natural estrus. Mean interval from calving to AI was 113 + 46 d. Only cows inseminated after 70 d were used in our study to avoid interference with bPSPB found in peripheral circulation during the postpartum period (18). Heifers were systematically inseminated at 72 and 96 h after the second of two 25-w doses of prostaglandin (tromethamine salt) administrered by intramuscular (1-m.) injections (Dynolytic ND, Upjohn, France) at 12-d intervals. Among these 145 animals, 17 cows and nine heifers were used for this experiment after two successive AIs and two additional cows were used after three AIs, resulting in a total of 175 sampling sequences following AI. Samples and Assay Procedures Early pregnancy diagnosis was performed by P4 RIA from plasma samples 24 d post AI. Additionaly, the females were bled at 24, 26 and between 30 and 35 d post AI for the bPSPB RIA. Palpation per rectum was performed at 70 t 9 d post AI (n = 120) to confirm pregnancy in cows or heifers that had not been observed in estrus. When diagnosis was questionable cows were rectally examined again, 10 to 20 d later. Blood (15 ml) was collected from the jugular vein into heparinized

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vacutainers (Beckton Dikinson, France). Plasma was separated by centrifugation and stored at -20°C until assays were performed. An assay procedure, previously described and validated by Thibier and Saumande (191, was used to determine P4 concentrations from single aliguots of plasma. Antiserum 111/6 (specific antisera LTD 1981 U.K.) raised in a goat against progesterone 11~ succinyl BSA was used at a final dilution of 1:12000. The assay sensitivity was estimated at 0.05 ng/ml. Inter- and intra-assay coefficients of variation (CVs) were 6.8 and 5.9% when duplicate estimates where run in the assay (n = 7) for a 1.5 ng/ml (1.47 + 0.03) reference plasma sample. Coefficients were 7 and 5.%, respectively, when duplicate estimates were run (8 assays) for a 4 ng/ml (4.27 f 0.09) sample of reference plasma. Validation criteria for the bPSPB RIA have been already described by Sasser et al. (17). Assays were performed on duplicate plasma aliguots (2OCpl each). Rabbit antiserum to bPSPB (RGS 38-l) was used at a final dilLtion of 1:305,000. It has previously been shown that the assay was bPSPB-specific, with minimal cross-reactivity (<0.05%) with bovine luteinizing hormone (LH) and ovine follicle stimulating hormone (FSH) and no reactivity with other hormones such as bovine prolactin (bPRL), bovine thyroid stimulating hormone (bTSH) or bovine growth hormone (bGH1. The limit of assay detection was estimated at 135 pg/assay tube (preparation R-37). Intra- and inter-assay CVs were 6.8 f 0.9% and 20.4.+ 7%, respectively. Data Analysis Cows and heifers were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB assay, at each stage after AI, cows were declared nonpregnant when at least one of the B/Be x 100 replicates was higher than 95%. Accuracy of pregnancy detection was calculated by comparing results of each RIA with results of palpations per rectum. At each stage after AI, accuracy of positive results refers to the number of animals tested positive and pregnant by rectal examination (numerator) among those tested positive (denominator) either by P4 or bPSPB assay. Similarly, accuracy of negative results refers to the number of animals tested negative and nonpregnant (numerator) among those tested negative by the assay (denominator). Efficiency of the detection of nonpregnant females refers to the number of animals detected nonpregnant by a given test (numerator) compared with the total number of animals found nonpregnant at the end of the experiment (denominator). The distribution of individual observations according to the PSPB results at each day of sampling, the type of animal and pregnancy status at the end of the experiment is presented (Table 1). These data were used to calculate the percentages of accuracy and efficiency presented in cells of subsequent Tables 2 and 4.

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Table 1. Distribution of the 175 observations according to pregnancy specific protein B results each day of sampling, type of animal (heifer or cow) and pregnancy status at the end of the experiment PSPB Day 24

Results

Day 26

_ + + + + + + Total

Days 30 to 35

+ +

+ + +

+

Animals h Heifers NP P

cows P NP

0 3 0 10

34e 1 lf 1

0 7 0 13

0 0 0 28

0 0 0 0

o 0 0 22a

41

37'3

42

46 0 0 1 1s 0 1c 6h 55

Total P NP

Total

80 1 1 2

80 11 1 25

o 0 0 50

1 0 1 6

1 0 1 56

83

92

175

0 10 0 23

hP= pregnant; NP = nonpregnant. For example in Table 2,when calculating accuracy of positive bPSPB results at Day 24 in cows (22/30),22 is given by a in the last row and 30 is equal to a + b + c + d in the same square in the lower part of the table. In Table 4,when calculating the efficiency in the detection of nonpregnant heifers at Day 30 to 35 (35/37), 35 is given by e + f and 37 by g . Accuracy and efficiency of the different techniques or sampling periods in the detection of pregnant and nonpregnant animals were subsequently compared by Chi-square tests. RESULTS When the experiment was completed following 175 AIs, 42 cows and 41 heifers were declared pregnant by palpation per rectum (Table 1) for a total conception rate of 47.4% (83/175). The pregnancy rate was not significantly higher in heifers than in cows (52.5 vs 43.3%; P> 0.05). Accuracy of early pregnancy diagnosis based on P4 concentrations at Day 24 is reported in Table 2. Data were not significantly different between cows and heifers for both positive and negative diagnoses. Accuracy of positive results at Day 24 was 67.2%. of the 40 females incorrectly diagnosed positive by the P4 assay at Day 24, 31 were found positive when data from the P4 assay at Day 24 were combined with assay results of Day 22 (data not shown). These animals likely had a delay in luteolysis. The nine other animals had ovulations followed by a rise in P4 concentration prior to Day 24. Accuracy of negative results for heifers and cows was always close or equal to 100%.

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I-HERIOGENOLOGY

The accuracy rates of pregnancy diagnoses based on the results of the bPSPB assay, at different stages of pregnancy are also illustrated in Table 2. At 24 and 26 d of pregnancy, the accuracy of positive results was higher in heifers than in cows. All heifers diagnosed pregnant as early as 24 d after AI maintained pregnancy; only 13% of the cows did. From Day 24 to Days 30 to 35, the number of animals diagnosed positive with the bPSP0 assay varied from 58 to 92. Among pregnant cows (Table 1) 60% (W/83), showed a rise in hPSP6 concentrations at 24 d. This indicates that in pregnant cows the moment when the first bPSPD elevation can be detected is highly variable. The proportion of inaccurate positive diagnoses decreased as gestation age increased (P< 0.05), and at the time of palpation, only one heifer with a positive bPSPB RIA was found to be nonpregnant. Table 3 shows the distribution of inaccurate positive PSPB results according to the sampling day of the 12 females Incorrectly diagnosed; 10 had posltlve plasma P4 at 24 d. In 83% of the cases the presence of bPSPB was associated with maintenance of corpus luteum function at or after 24 d. But of the 31 cows with inaccurate positive P4 on Days 22 and 24, only 10 had detectable bPSPB (32.3%). This shows that only one third of the animals with a delayed luteolysls had a rise in bPSPB. Table 3. Number of animals with positive serum pregnancy specific protein B but found nonpregnant by palpation per rectum distributed according to different sampling days and time of luteolysis Pregnancy Speclflc Protein B Results Day 24

+ +

Day 26

+ + + •t

Day 30 to 35

+ +

No.of animals

Palpation per rectum

5 1 1

6 1 1 1 1 1 1

1 1 1

12

10

+

f

+ + + Total

No with luteolysis after 24 da

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By the end of this experiment conception had not occurred after 92 AIs. Reliability in detecting nonpregnancy is critical. Table 4 shows that the efficiency of detecting nonpregnancy regardless of assay type or time of sampling after AI, was not different between heifers or cows. The percentage of nonpregnant females detected by the P4 RIA at 24 d was 56.5%. This percentage was considerably greater (P< 0.001) when the bPSPB assay was used. At 26 or 30 to 35 days post AI, more than 89% of the nonpregnant females were detected. Table 4. Efficiency of detection of nonpregnant cows and heifers by progesterone or pregnancy specific protein B radioimmunoassay at various days after artificial insemination

Tyrx

of

Progesterone

Pregnancy specific protein B

animal Day 24 % No.

Day 26 % No.

Day 30 to 45 % No.

cow Heifer

65.0 43.2

(36/55) (16/37)

85.0 (47/55) 94.6 (35/37)

87.0 (48/55) 95.0 (35/37)

Total

56.5

(52/92ja

89.1 (82/92)b

90.2 (83/92)c

a vs b vs c, P< 0.001. a vs b , P< 0.001. DISCUSSION Accuracy of positive diagnoses by P4 RIA at 24 d was lower than 70%..This data is similar to that obtained from a large .~ number of cattle in the same laboratory (8) and by other authors (7,20,21). However,these percentages of accuracy are lower than those reported by Heap et al. (61, Van de Wiel et al. (22), Booth (231, Booth et al. (4) Laing et al. (24), Gowan et al. (25) and Inaudi et al. (26). This may have been due to using cows that had been inseminated one or two times before being included in the experiment and were perhaps a subfertile group. Actually, Thibier and Terqui (27) and Humblot and Thibier (8) reported that the accuracy of positive tests is highly related to this factor. Accuracy of negative tests was nearly lOO%, as is usually reported in P4 RIAs (4,23,26,28,29) and EIAs (14,30,31). At the end of the experiment, the percentage of correctly detected nonoregnant females by the progesterone assay from samples collected at 24 Eddid not exceed 60%. This can be attributed mainly to embryonic mortality occurring after Day 16 and followed by late luteolysis. Such events have already been described by Kummerfeld et al. (32) and Humblot and Dalla Porta (33). The bPSPB assay was more efficient than the progesterone assay in the detection of nonpregnant females, since only 25% of those with

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a positive progesterone assay at Day 24 had an elevated bPSPB concentration. This study further shows that an early positive PSPB test that did not detect pregnancy was strongly associated with the maintenance of the corpus luteum longer than 24 d. Most of the cows in this condition were PSPB positive in two or more successive samples. This diminishes the possibility of assay error for these cows and further suggests that late embryonic mortality after Day 16 is the major reason for an inaccurate diagnosis of pregnancy by the bPSPB assay. Despite this, the accuracy of a positive bPSPB test was less affected by late embryonic mortality than the progesterone test, since only one third of the cows with a delayed luteolysis (positive serum progesterone at both 22 and 24 d post AI) had detectable serum PSPB. Moreover, when measuring progesterone concentrations the day of AI, it was shown that in French conditions, 5 to 10% of cows may be inseminated at the wrong time, as when progesterone concentrations in plasma are higher than 1.5 ng/ml (34,35). Cows inseminated during estrus are likely to be positive 24 d later and may bias estimates of embryonic death based on progesterone assay. This could have contributed to lowering the accuracy of the progesterone positive results but not the accuracy of bPSPB positive results. Finally, the PSPB assay allowed the detection of about 90% of nonpregnant females between Days 26 and 30 to 35 after AI. These data and the results obtained at time of palpation are very similar to those reported by Mauer et al. (36) and Sasser et al. (17) for pregnancy detection by bPSPB. Accuracy of negative diagnosis did not approach 100% before Days 30 to 35 after AI. This was due to the appearance of bPSPB later than 26 d in the plasma of several pregnant cows, and it suggests that the test is most effective after 26 d in dairy cows. Accuracy of a positive bPSPB test was always higher than 85%. The 100% accuracy rate reported in heifers at Day 24 shows that females having an early rise in bPSPB concentration in plasma remained pregnant. Moreover, greater accuracy of positive detection in heifers showed that late embryonic mortality associated with elevated bPSPB levels occurred more frequently in cows. This is consistent with earlier reports showing an increase in the frequency of late embryonic mortality due to age (37,38). It can be concluded from these data that in dairy cows when AI is performed after 70 days postpartum, at 30 d post AI or later, the the bPSPB assay is a very efficient test for both pregnancy prediction and detection of nonpregnant females. REFERENCES

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14. Van de Wiel, D.F.M. and Koops, W.K. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma. Anim. Reprod. Sci. g:201-213 (1986). 15. Reimers, T-J., Ruder, C.A. and Sasser , R.G. Production of pregnancyspecific protein B (bPSPB) by bovine binucleate trophoblastic cells. Biol. Reprod. -32 (Suppl. 11):65 abstr. (1985). 16. Butler, J.E., Hamilton, W.C., Sasser, R-G., Ruder, C.A. Hass, G.M. and Williams, R.J. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod..-26: 925-933 (1982). 17. Sasser, R.G., Ruder, C.A., Ivani, K.A., Butler, J.E. and Hamilton W.C. Detection of pregnancy by radioimmunoassay of a novel pregnancyspecific protein in serum of cows and a profile of serum concentrations during gestation. Biol. Reprcd. -35: 936-942 (1986). 18. Ruder, C.A. and Sasser, R.G. Source of bovine pregnancy-specific protein B (bPSPB) during the postpartum period and estimation of halflife of bPSPB. Annual meeting Am. Sot. Anim. Sci. J. Anim. Sci. -63 (Suppl.1): 335 (1986). 19. Thibier, M. and Saumande, J. Oestradiol 178 , progesterone and 17clhydroxyprcgesterone concentration in jugular venous plasma in cows prior to and during oestrus. J. Steroid Biochem. 5:1433-1437 (1975). 20. Cox, N.G., Thompson, F.N. and Culver, D.H. Milk progesterone to predic reproductive status in a commercial dairy herd. J. Dairy Sci. -61: 1616-1621 (1978). 21. Gowan, E.W. and Etches, R.J. A solid phase radioimmunoassay for progesterone and its application to pregnancy diagnosis in the cow. Theriogenology -12:327-343 (1979). 22. Van de Wiel, D.F.M. and Koops, W.K. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blocd plasma. Anim. Reprod. Sci. -10:201-213 (1986). 23. Booth, J.M. Milk progesterone testing: application to herd management. J. Dairy Sci. -62:1829-1834 (1979). 24. Laing, J.A., Gibb, H.A. and Eastman, S.A. A herd test for pregnancy in cattle based on progesterone levels in milk. Br. Vet. J. -136:413-415 (1980). 25. Gowan, E.W., Etches, R.J., Eryden, C. and King, G.J. Factors affecting accuracy of pregnancy diagnosis in cattle. J. Dairy Sci. -65: 1294-1302 (1982). 26. Inaudi, P., Bacigalupo, M., Monittola, C., Lugaro, G. and Gennazzani, A.R. Pregnancy diagnosis in cattle by a rapid and highly reliable method for progesterone determination in milk. J. Reprod. Fertil. 65: 265-273 (1982).

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27. Thibier, M. and Tergui, M. Les diagnostics de gestation et de non gestation chez les mammifkres domestigues de ferme. Collogue de la Ste Francaise d'etude de la sterilite et de la fertilite. Masson, Paris, 1978, pp.127-146. 28. Calrestany,D. and Foote, R.H. The use of milk progesterone and electronic vaginal probes as aids in large dairy herd reproduction management. Cornell Vet. -75:441-453 (1985). 29. Thi.bier,M., Petit, M. and Humblot, P. Use of progesterone concentrations in peripheral plasma or milk in cattle herd management. In: Control of Reproduction in the Cow. Martinus Nijhoff, The Ague, 1977, pp. 576-595. 30. Chang, C.F. and Estergreen, V.L. Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows. Steroids -41:173-195 (1983). 31. Sauer, J.M., Foulkes, J.A., Worsfold, A. and Morris B.A. Use of progesterone II-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre plate enzyme immunoassay of progesterone in milk and its application to pregnancy testing in dairy cattle. J. Reprod. Ferti1.76:375-391 (1986). 32. Kummerfeld, H.L., Oltenacu, E.A.B and Foote, R.H. Embryonic mortality in dairy cows estimated by non return to service, estrus and cyclic milk progesterone patterns. J. Dairy Sci. -61:1773-1777 (1'382). 33. Humblot, P. and Dalla Porta, M.A. Effect of conceptus removal and intrauterine administration of conceptus tissue on luteal function in the cow. Reprcd. Nutr. Dev. -24:529-541 (1984). 34. Thibier, M. and Rakotonanahary, A. Concentration de la progesterone plasmatigue lors de 1'IA et taux de fertilitd chez la vache laitikre El. Insem. 159:3-10 (1977). 35. Hunblot, P., Oliveira, O., Ruder, C., Jeanguyot, N., Thibier, M. and Sasser, G. Progesterone and PSPB concentration at time of AI and at the beginning of pregnancy in the dairy cow. In: Proceedings of A Bard Workshop on Maternal Recognition of Pregnancy and Maintenance of the Corpus Luteum. Jerusalem, Israel. 62 abstr. (1988). 36. Mauer, R.R., Ruder, C.A. and Sasser, R.G. Effectiveness of the protein B radioimmunoassay to diagnose pregnancy in beef cattle. J. Anim. Sci. -61 (Suppl.l):390 abstr. (1985). 37. Ball, P.J.H. The relationship of age and stage of gestation to the evidence of embryo death in dairy cattle. Res. Vet. Sci. -25 120-122 (1978). 38. Hunblot, P. and Denis, J.B. Sire effects on cow fertility and late embryonic mortality in the Montbeliard breed. Lives. Pro. Sci. -14: 139-148 (1986).

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