A solid-phase radioimmunoassay for progesterone and its application to pregnancy diagnosis in the cow

A solid-phase radioimmunoassay for progesterone and its application to pregnancy diagnosis in the cow

THERIOCENOLOGY A SOLID-PHASE RADIOIMMUNOASSAY FOR PROGESTERONE AND ITS APPLICATION TO PREGNANCY DIAGNOSIS IN THF. COW E. W. Gowan R. J. Etches Depart...

833KB Sizes 0 Downloads 34 Views

THERIOCENOLOGY

A SOLID-PHASE RADIOIMMUNOASSAY FOR PROGESTERONE AND ITS APPLICATION TO PREGNANCY DIAGNOSIS IN THF. COW E. W. Gowan R. J. Etches Department of Animal and Poultry University of Guelph Guelph, Ontario NlG 2W1 Received

for publication:

August

Science

,9,1,7,979

ABSTRACT A solid-phase radioimmunoassay using antibody-coated tubes and 125I_ progesterone label was developed to provide an alternative to the conventional aqueous method of assaying progesterone in milk for early pregnancy diagnosis. The accuracy of diagnoses made using the solid-phase assay of progesterone in milk was assessed by comparison with diagnoses made using an aqueous assay of serum progesterone. Both methods agreed in When "fat-free" milk was assayed the thirteen cows that were diagnosed. by both aqueous (x) and solid-phase (y) methods, the progesterone values which resulted showed a high correlation (r = .94) and a linear relntionship of y = 1.67x - 0.68. MilK samples, in which the fat cxcentration ranged from 0.20 to 4.04%, were assayed by the solid-phase method and nc relationship between milk fat and progesterone concentration was observed. Pregnancy diagnosis from solid-phase assay of milk samples collected on the day of breeding, 21 and 23 days following breeding was performed on 62 Holstein cattle. The accuracy of "non-pregnant" diagnoses was 95% to 100% and the accuracy of "pregnant" diagnoses was 80% if breeding day values were used and 72% if these vaiues were excluded. The accuracy of this assay in diagnosing pregnancy was equal to that of previously published assays and provided the advantages of requiring In addition, foremilk, composite less technical time and equipment. or stripping samples can be accomodated in this assay since the estimates of progesterone are not affected by the concentration of milk fat. INTRODUCTION One of the most important goals of the modern dairy farmer in his efforts to maximise reproductive potential of his COWS is to achieve The gestation period of the bovine, ranga 12 month calving interval. ing from 277 to 290 days in length, allows the herdsman only 75 to 100 days following parturition in which to reimpregnate the cow if this goal This research project was funded by Agriculture Canada Acknowledgements: The authors wish to thank Richard Buck and the contract 01845-O-1265. employees at the Elora Dairy Research Centre for collecting blood and The milk samples, and Dr. G. J. King for performing an ovariectomy. supply of 1251-proges:erone and antibody-coated tubes from Micromedic Systems is also gratefully acknowledged.

DECEMBER

1979 VOL. 12 NO. 6

3’7

THERIOGENOLOGY If the cow is first inseminated 50 to 60 days post is to be realised. partum there are a maximum of 3 estrous cycles for pregnancy to become Failure to achieve a U-month calving interval represents established. significant economic loss of milk and progeny per cow or herd per year. An early test for pregnancy which is based on the cycle nature of progesterone production during the bovine estrous cycle, has been developed for cattle. This test measures the quantity of progesterone in milk or plasma and can be performed 21 to 24 days following insemination (1-3). The "not-pregnant" diagnosis can be routinely performed with 90 The high degree of accuracy associated with to 100% accuracy (1, 4-8). this diagnosis is the most valuable feature of this test. Dairymen can confidently take appropriate action toward cattle which do not conceive and recognize cases of subfertility much sooner than would be possible if only rectal pregnancy diagnosis at 40 to 60 days following breeding is practised. Unfortunately, only 70 to 80% accuracy can be achieved when making a "pregnant" diagnosis (1, 4-9). The error associated with positive diagnoses, which has been attributed to early embryonic death, makes rectal palpation of cattle 40 to 60 days following breeding advisable to confirm the presence of a developing fetus. The progesterone test may serve as a diagnostic tool for veterinarians by providing useful information on reproductive cycles of 'problem' cows and help monitor the effects of treatment of these cattle. Progesterone analysis of milk samples could be a very informative addition to the herd health programs offered by many veterinary practices. This paper reports the development of a solid-phase radioimmunoassay for progesterone which is more suitable for clinical application than the conventional aqueous-phase method of analyzing milk samples. The solid-phase method uses polypropylene tubes which are coated with an antiserum raised against progesterone and utilises 1251-progesterone as the radioactive tracer. The solid-phase assay is compared with the aqueous method for diagnosing pregnancy and non-pregnancy, and for its adaptability to automation in order to accommodate the demands of a clinical pregnancy diagnostic service. MATERIALS The solid-phase

125

I-progesterone

AND METHODS

assay

The solid-phase assay was performed in 0.8 x 5 cm polypropylene tubes which were coated with antibody raised in rabbits against pro esterone llcc-succinyl-bovine serum albumen. The label was progesterone-llor- ?25Ityrosine-methyl-ester. Both the label and antibody-coated tubes were generously provided by Micromedic Systems Inc., Horsham, Pennsylvania. To each reference tube, the following were added: 0.6 ml of gelatinphosphate buffer (0.1 M potassium phosphate, 0.1% gelatin (Fisher, Granular Laboratory Grade), 0.01% sodium azide, pH 7.0); 0.1 ml of casein buffer (1.25% (w/v) casein (Fisher, Certified Vitamin-Free) in 0.1 M potassium phosphate, 0.01% sodium aside, pH 7.0); and 0.1 ml of gelatin-phosphate buffer containing between 1.6 ng and 6.25 pg of progesterone (Sigma Chemical Co., St. Louis, Missouri). Each sample tube received 0.7 ml of gelatin-phosphate buffer and 0.1 ml of a milk sample. All tubes received

328

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY

0.1 ml of gelatin-phos hate buffer containing approximately 15,000 cpm of progesterone-lla- 12'1-tyrosine-methyl ester. To assess the effects of time and temgerature on thg assay, tubes containing label only were incubated at 37 C, 22OC. or 4 C for 1, 2, 4, 8 or 18h. Routinely, the buffers, standards or samples, and label were added sequentially, the tubes were shaken for 1 set and the assay was incubated at 22OC for 5h or overnight. At the end of incubation the contents were discarded and 0.5 ml of gelatin-phosphate buffer was addedl;? each tube. This buffer was decanted within 5 min and the amount of I-progesterone bound to the tube was quantitated in the Searle 1185 scintillation spectrometer. The effect of the protein milieu of milk on the binding of the label to the antiserum was tested by adding to each standard tube 0.1 ml of milk for an ovariectomized cow, 0.1 ml of milk from a cow in heat from which endogenous progesterone was removed by adding 2.5 mg charcoal (Norite A neutral, Fisher)/ml of milk, incubating at 37OC for 30 min and centrifuging at 10,000 for 30 min to remove the charcoal, or 0.1 ml of the 1.25% casein buffer. The cross-reactions of the solid-phase assay were assessed by substituting various steroids for the progesterone standards and comparing the masses of hormone required to displace 50% of the label. The amount of progesterone in a sample was calculated by expressing thedisplacementas a percentage of the 0 standard and manually interpolating this value from a linear-logarithmic plot of % bound vs mass of progesterone. Preparation

of samples

A 30 ml composite sample of milk was collected in the morning and stored at 4'C after the addition of potassium dichromate tablet (Arnold Nasco, Guelph). Prior to assaying, the milk fat was allowed to separate by sedimentation for 24 hours. The surface fat layer was aspirated from each sample and discarded. The remaining portion of each sample was then assayed for progesterone. To test the efficiency of this procedure, milk was allowed to sediment for 0, 12, 24 and 50 hours, the fat layer was removed and the amount of progesterone was quantitated in the remaining milk. To obtain relative changes in milk fat, the height of the fat layer was measured and expressed as a percentage of the total height of the milk sample. The absolute fat content was measured by the infra-red milk analysis (IRMA) method (10). In an attempt to remove more of the milk fat, the samples were centrifuged at 600 g for 20 min, the fat layer was aspirated and the remaining 'fat-free' milk was assayed for progesterone. In preliminary tests, blood and milk samples were collected on the same day. The blood was stored at 4OC for 24 hours and then centrifuged at 600 g for 20 min. The serum was collected and frozen at -2OOC until assayed for progesterone in an aqueous assay. The aqueous progesterone

assay

To validate the use of the solid-phase assay for quantitating progesterone in milk and for diagnosing pregnancy, various samples of blood serum and

DECEMBER

1979 VOL. 12 NO. 6

329

THERIOGENOLOGY of centrifuged milk were assayed either by the solid-phase method or by an aqueous assay using a (1,2,6,7-3H)progesterone label. Two tests Firstly, milk with the aqueous progesterone assay were performed. samples that were assayed by the solid-phase assay were centrifuged, the fat layer was aspirated and the sample was assayed with the aqueous assay. The correlation between the two estimates and the regression equation describing the relationship between the two methods was calculated. Secondly, serum concentrations of progesterone were estimated by the aqueous assay and diagnoses of pregnancy were made according to the relative values on the day of breeding and 21 days later. Milk samples collected on corresponding days from the same cows were assayed by the solid-phase method and a second diagnosis of pregnancy was made. The two diagnoses were then compared with each other and with that of a veterinarian after rectal palpation of the reproductive tract between 45 and 60 days after breeding. The procedure for the aqueous assay was a slightly modified version of the method reported by Furr (10). The modifications included two extractions of 0.5 ml of milk or serum with 3 ml of iso-octane and the use of an antiserum raised in a rabbit against progesterone-Ila-succinylThe cross-reactions bovine serum albumen (Steraloids, Wilton, N.H.). of the antiserum with cholesterol, testosterone, estradiol, co;tisol, corticosterone, deoxycorticosterone 17a-OH-progesterone, pregnenolone, 5n-pregnsn-3a-ol-ZO-one, 4-pregnen-ZOB-ol-3-one, 58-pregnan-3cr-01-20one, 5!3-pregnan-38-ol-20-one, 5a-pregnan-3B-ol-20-one, and 4-pregnen20a-ol-3-one, were less than 1%. The cross-reactions with ll,+OH-progesterone, 50-pregnan-3,20-dione and 5a-pregnan-3,20-dione were 5.4%, 6.7% and 15.6%, respectively. Collection

of samples

for pregnancy

diagnosis

Blood and/or milk was collected on the day of breeding, 21 days and 23 days after breeding from 62 Holstein cattle. These samples were used to diagnose pregnancy (1,12). Cattle which were bred more than once provided more than one diagnosis. A final diagnosis of pregnancy was made by palpating the uterus during a rectal examination performed 45 to 60 days after breeding. All samples were obtained from cows in the University of Guelph Dairy Research Unit at Elora, Ontario. RESULTS The solid-phase

125

I-progesterone

assay

The standard curve for the solid-phase assay is shown in Fig. 1. The sensitivity of the assay was 6.25 pg and the useful range of the standards extended to 1.6 ng. The inclusion of 0.1 ml milk from an ovariectomized cow, 0.1 ml of 1.25% casein buffer or 0.1 ml of progesterone-free milk decreased the binding throughout the standard curve by 8 to 15% (Fig. 1). The binding could be increased by approximately 5% if the final addition and removal of 0.5 ml of gelatin-phosphate buffer was omitted. The effects of varying time and temperature are shown in Fig. 2. Incubation at 37'C resulted in a more rapid equilibration than at either 22OC or 4OC. After 18h of incubation at 4oC, the binding did not equal

330

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY

that observed

after 4h at 22' at 22' or 37'C.

The cross-reactions of the assay were less than 1% with testosterone, estradiol, cortisol, corticosterone, 17ti-hydroxy-progesterone, pregnenolone, 4-pregnen-ZOB-ol-3-one, 5E-pregnan-3a-ol-20-one, and 4pregnen-ZOol-ol-3-one. The cross-reaction with deoxycorticosterone, 51pregnan-313-ol-20-one, 5u-pregnan-3B-ol-20-one, 5I?-pregnan-3,20-dione, llB-hydroxy-progesterone and 5n-pregnan-3,20-dione was 1.14, 1.28, 1.67, 6.40, 6.40 and 8.90%, respectively. The within-assay coefficient of variation for samples containing between 0.10 and 0.50 "g/ml and more than 2 "g/ml was 6.02% and 10.84"/, respectively. The between-assay coefficient of variation of the 25 pg and 0.1 ng standards was 13.0 and 16.9%. respectively. Coefficients of variation were calculated according to the method described by Kodbard (13). Preparation

of samples

Sedimentation of the milk samples for up to 50 hours resulted in .I progressive decline in the amount of fat remaining in the milk sample (Fig. 3). When the amount of fat remaining in the sample was expressed as a percentage of the total amount of fat, there was no difference between the "IRMA" and "linear" methods of calculating the fat concentration. In absolute values, the mean concentration of fat at time 0 was 3.04% (ranging from 1.62% to 4.04%) and after 50 hours of aedimentation only 0.63% (ranging from 0.20% to 1.16%) remained. In two separate trials, the concentrations of progesterone were unaltered in the milk remaining after 0, 12, 24 and 50 hours of sedimentation (Table I). Analyses of variance of the samples from the "LRHA" trials, the "linear' trial and the combined trials indicated that the mean concentrations of progesterone were not significantly changed aftisr up to 50 hours of sedimentation (F < .05; P ,. .99). Similarly, centrifuging the milk samples in the 'linear' trial (Table I) did not significantly (F = .03, p j .99) alter the concentration of progesterone. Validation

of the

125

I-progesterone

assay for pregnancy

diaeis

The correlation between the concentration of progesterone in "fatfree" milk measured by the aqueous and solid-phase assays was 0.94. '['hk' relationship between the amount_of progesterone assayed by the solidphase (y) and aqueous (x) methods was best described by the equation y = 1.67x - 0.68. The slope of this equation was significantly different from the expected value of one (t = 4.46, P > 0.01). In the thirteen cows from which serum and milk were obtained, pregnancy could be diagnosed with equal accuracy using either fluid (Table TI). This comparison was made using the 1251-progesterone solid-phase assay to quantitate progesterone in milk and the aqueous assay to quantitatr progesterone in serum. The absolute values were not comparable, although the relative differences between the amounts of progesterone on the day of breeding and 21 days later did not allow a valid comparison of the abilir\* of the two methods to diagnose pregnancy. Using the aqueous assay, an abnormally high concentration of progesterone was observed on day 0 in the serum from cow 854. An explanation for this is not readily apparent since milk removed at tht-

DECEMBER

1979 VOL. 12 NO. 6

331

THERIOGENOLOGY same time from this cow contained normal amounts of progesterone. Pregnancy

diagnosis

A histogram (Fig. 4) of 333 observations of the concentration of progesterone on the day of breeding, and on 21 and 23 days after breeding revealed two peaks in the values obtained. No values were Therefore, the arbitrary decision obtained between 1.01 and 1.10 ng/ml. was made to designate all cows whose milk contained less than 1.01 ng/ml on days 21 or 23 after breeding as non-pregnant. Conversely, if the 21 or 23-day sample contained more than 1.01 ng of progesterone/ml, the cow was diagnosed pregnant. Using these criteria, the accuracy of the assay is presented in Table III. Regardless of the parameters or combinations of parameters chosen to diagnose pregnancy, 94 to 100% accuracy was achieved for the negative diagnosis. If the stipulation that the breeding-day sample must contain less than 1 ng/ml was made, then approximately 80% of the positive diagnoses were confirmed by rectal palpation of the uterus. If diagnoses were made after consideration of only the 21 or 23-day samples, 71.7% and 73.2%, respectively, of the positive diagnoses were subsequently confirmed. Although the use of a breeding-day value increased the accuracy of positive diagnoses, it created a substantial class of "questionable" diagnoses. In these cases both the breeding-day sample and the 21 or 23-day sample contained more than 1.01 ng of progesterone/ml of milk. When the samples from questionable diagnoses were reassayed, approximately 50% of these enigmatic situations were resolved within the limits of the arbitrary pregnant and non-pregnant classifications (Table III). The mean concentrations and the range of concentrations of progesterone in samples taken on days 0, 21 and 23 are presented for the pregnant and non-pregnant classifications in Table IV. DISCUSSION lbe solid-phase

progesterone

assay

The assay was shown to be accurate, specific, repeatable and sufficiently sensitive to measure progesterone in milk from cows. For clinical applications, the assay offers the advantages of eliminating the need for a centrifuge, eliminating the need for expensive scintillation cocktails which are associated with B counting and increasing the sensitivity in comparison to most aqueous assays. In addition, the assay could be simplified so that only two additions of reagents are required per tube. Although washing the tubes at the end of the assay reduces the binding by approximately 5%, the final estimates of concentration were not altered since this procedure was applied to both sample and standard tubes. Therefore, in manual operations, the final washing of tubes could be eliminated without a loss of accuracy. The inclusion of 0.1 ml of milk known to be free of endogenous progesterone severely reduced the binding of the label to the antiserum and this effect was not parallel throughout the assay (Fig. 1). Therefore, some correction factor must be applied to obtain approximately absolute values. Milk from cows in heat, which is known to contain low concentrations of progesterone, can be used for this purpose after treat-

332

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY

ment with charcoal (Fig. 1). Alternatively, 0.1 ml of a 1.25% casein buffer can be substituted for milk from ovariectomized cows. The simplicity of this latter method makes it the method of choice for correcting the errors in estimation which are introduced by using an unextracted milk sample. For reasons of technical convenience, assays were routinely incubated with the antibody for 5h or overnight at 22'C. However, by incubating the assay at 37'C, incubation time can be shortened to 2h. Using this faster procedure, it is possible to complete one assay containing 400 tubes in one working day. Using aqueous assays of progesterone, Dobson and Fitzpatrick (4) and Pope St. (14) reported correlations of 0.88 and 0.99 between the concentrations of fat and progesterone in milk samples. In this particular assay, however, the amount of fat in the sample had no significant effect on the estimate of progesterone concentration (Table I and Fig. 3). It should be noted that the greatest fat concentration which was observed was 4.04% and that the mean concentration was only 3.04%. In the Elora Dairy Research Unit, the average fat concentration in the herd during the sample period was 3.45%, a value which is closer to the breed-classaverage for Holsteins in Ontario. In part, the low values observed in the samples included in the study were attributed to withdrawal of milk samples from the morning milking. In a more extensive diagnostic service, samples containing much larger amounts of fat would be anticipated. The potential problem that these samples might introduce can be prevented by sedimenting the milk for 24 hours. From the data in Fig. 3, it is apparent that 62% of the fat in the sample is removed by this procedure. Therefore, samples containing up to 8% fat can be accommodated in this assay and it is possible that even greater levels may not affect the accuracy of the assay. Shelford -et al. (15) reported mean values between 1.36 and 1.81, 3.45 and 3.85, and 8.17 and 8.88% fat for foremilk, composite and stripping samples, respectively. Therefore, all foremilk and composite samples would be suitable for this assay and it is likely that most stripping samples could also be assayed with confidence. Validation

of the

125

I-progesterone

assay

The high correlation between the amount of progesterone in the milk sample when assayed by either the solid-phase or aqueous methods indicated a close linear relationship between these methods. However, the slope of the regression equation was significantly different from the expected value of one and the intercept of this line was negative. Relative to the aqueous assay, therefore, the solid-phase assay yields lower estimates of milk progesterone below 1 ng/ml whereas above 1 ng, the solid-phase assay yields higher estimates of progesterone. These differences render comparisons of values between laboratories invalid but improve the relative comparisons upon which pregnancy diagnoses are made since the range of observations is increased (see Table IV). Robertson and Sarda (12) Shemesh -et al. (16) and Heap et al. (1) have used serum concentrations of progesterone to diagnose pregnancy in cows. From the data in Table II, it is apparent that the diagnoses made using the solid-phase assay for milk samples yielded results that were identical to those obtained using the aqueous assay for serum samples. The data in Table II indicated that milk was an acceptable substitute for serum and that the solid-phase assay was an acceptable substitute for the

DECEMBER

1979 VOL. 12 NO. 6

333

THERIOGENOLOGY aqueous

assay.

ed by others Pregnancy

The former conclusion (1, 14, 17, 18).

agrees with that previously

report-

diagnosis

The distribution in Fig. 4 emphasizes that the estimates of progesterone in milk on day 0, 21 and 23 form a continuum with poorly demarcated limits between the values in pregnant and non-pregnant cows. However, the decision to use 1 ng/ml as the upper limit for non-pregnant cows and the lower limit for pregnant cows allowed the elimination of a doubtful category without detracting from the accuracy of the assay. The accuracy of negative diagnoses is similar to that reported by Heap -et al. (5), Pope -et al. (14), Booth and Holdworth (4) and Dobson and Fitzpatrick (19). Ninety-five to 100% of all diagnoses were correct and this attribute of the assay may be its greatest practical asset. When positive diagnoses were made using the breeding-day sample and the 21 or 23-day sample, the accuracy of positive diagnoses was approximately 80% and this success rate declined to approximately 72% if only the 21 or 23-day sample was used as the criterion for judgement. In similar studies using only milk samples collected 21 to 24 days after breeding, Heap -et ah. (S), Shemesh -et al. (8), Pope -et al. (14) and Hoffman et al. (7) have reported that approximately 80% of all positive diagnoses are confirmed within 60 days after breeding. The highest success rate of 90% was reported by Booth and Holdsworth (4) whereas Cox, Thompson and Culver (9) reported a lower success rate of 67%. The cause of the low success rate for positive diagnoses in the present study is not clear. It may be a peculiarity of the University herd or it may be caused by an error in the lower limit of progesterone concentrations in the pregnant category. Part of the lower success rate may be caused by not creating a 'doubtful' category from which samples were reassayed. In the present study, samples were reassayed only if the breeding-day sample and the 21 and 2fday samples contained more than 1 ng progesterone/ml. After reassaying these questionable estimates, approximately 5% of the errors were attributed to the assay and therefore reallocated to the pregnant or non-pregr.ant classification. The remaining 5% continued as questionable diagnoses. These results indicated that some false positive diagnoses are the result of spurious overestimates of the concentration of progesterone. However, the questionable group of diagnoses which remained suggested the existence of significant number of 'abnormal' cows, several cases of improperly timed insemination, cycle lengths which are significantly shorter or longer than average and/or misidentification of milk samples. Nevertheless, the success of this assay in identifying pregnant animals was similar to that reported by several other authors (1, 4-9) and, although this assay will not decrease the frequency of false positive diagnoses, it requires less technical time and a smaller capital investment in equipment. Moreover, it is unaffected by the concentration of milk fat. Therefore, the use of this assay obviates the need for restrictions upon the time of day or method of collecting milk samples by the dairyman and should reduce reagent cost to a minimum.

334

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY REFERENCES 1.

Heap, R.B., Gwyn, Mearle, Laing, J.A. and Walters, D.E. Pregnancy Diagnosis in Cows; Changes in Milk Progesterone Concentration During the Oestrous Cycle and Pregnancy Measured by Rapid Radioimmunoassay. J. Agric. Sci. -81: 151-157 (1973).

2.

Pennington, J.A., Spahr, S.L. and Lodge, J.R. Pregnancy Diagnosis J. Dairy Sci. in Dairy Cattle by Progesterone Concentration in Milk. -59: 1528-1531 (1976).

3.

Hoffman, B., Hamburger, R., Gunzler, D., Komdorfer, L., and Lohoff, H. Determination of Progesterone in Milk Applied for Pregnancy Diagnosis in the Cow. Thereiogenology, 2: 21-25 (1974).

4.

Booth, J.M., and Holdsworth, R.J. The Establishment and Operation of a Central Laboratory for Pregnancy Testing in Cows. Br. Vet. J. -132: 512-528 (1976).

5.

Heap, R.B., Holdsworth, R.J., Gadsby, J.E., Laing, J.A. and Walters, D.E. Pregnancy Diagnosis in the Cow from Milk Progesterone ConcenBr. Vet. J. _132: 445-463 (1976). tration.

6.

Pennington, J.A., Spahr, S.L. and Lodge, J.R. Factors Affecting Progesterone in Milk for Pregnancy Diagnosis in Dairy Cattle. Br. Vet. J. -132: 487-496 (1976).

7.

Hoffman, gesterone dological Br. Vet.

8.

Shemesh, M., Ayalon, N., Shalev, E., Nerya, A., Schindler, H., and Malguir, F. Milk Progesterone Measurement in Dairy Cows: Correlation with Estrus and Pregnancy Determination. Theriogenology 2: 343-350 (1978).

9.

Cox, Nancy M., Thompson, F.N. and Culver, D.H. Milk Progesterone to J. Dairy Predict Reproductive Status in a Commercial Dairy Herd. Sci. 61: 1616-1621 (1978).

B., Gunzler, O., Hamburger, R. and Schmidt, W. Milk Proas a Parameter for Fertility Control in Cattle: MethoApproaches and Present Status of Application in Germany. J. 132: 469-476 (1976).

J. Dairy Sci. -55: 650-651

(1972).

10.

Biggs, D.A.

11.

Furr, B.J.A. Radioimmunoassay of Progesterone in Peripheral Plasma of the Domestic Fowl in Various Physiological States and in FolliActa Endocr. -72: 89-100 (1973). cular Venous Plasma.

12.

Robertson, H.A. and Sarda, 1-R. Very Early Pregnancy Test for MamIts Application to the Cow, Ewe and Sow. J. Endocr. -49: mals: 407-419 (1971).

13.

Statistical Quality Control and Routine Data Rodbard, David. Processing For Radioimmunoassays the Immunoradiometric Assays. Clin. Chem. -20: 1255-1281 (1974).

DECEMBER

Infrared Milk Analyser.

1979 VOL. 12 NO. 6

J.

335

THERIOGENOLOGY

14.

Pope, G.S., Majzlik. I., Ball, P.J.H. and Leaver, J.D. Use of Progesterone Concentrations in Plasma and Milk in the Diagnosis of Br. Vet. .J. 132: 497-506 (1976). Pregnancy in Domestic Cattle.

15.

Shelford, J.A., Grisenthwaite, T., Barrington, S., Peterson, R.G. and Fisher, L.J. Milk Sampling Methods for Progesterone Assay for Can. J. Anim. Sci. -59: 77-82 (1979). Early Pregnancy Diagnosis.

16.

Shemesh, M., Ayalon, N. and Linder, H.R. Early Pregnancy Diagnosis Based Upon Plasma Progesterone Levels in the Cow and Ewe. 3. Anim. Sci. 36: 726-729 (1973).

17.

Ginther, O.J., Nuti, L., Wentworth, B.C. and Tyler, W.J. Progesterone Concentration in Milk and Blood During Pregnancy in Cows. Proc. Sot. Exp. Bio. Med. -146: 354-357 (1974).

18.

Dobson, H., Midmer, Sandie E. and Fitzpatrick, R.J. Relationship Between Progesterone Concentrations in Milk and Plasma During the Bovine Oestrous Cycle. Vet. Rec. 96: 222-223 (1975).

19.

Dobson, H. and Fitzpatrick, R.J. Clinical Application of the Progesterone in Milk Test. Br. Vet. J. 132: 538-542 (1976).

336

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY TABLE I - CONCENTRATION OF PROGESTERONE IN "FAT-FREE" MILK (ng/ml) PREPARED BY SEDIMENTATION FOR 0 TO 50 HOURS OR BY CENTRIFUGATION

Trial

Number of cows

IRMA

10

Linear Combined

10 20

0

12

2.45 2.89 2.67

2.41 2.85 2.63

Time 24

2.75 2.74 2.74

50

Centrifuged

S.E.M.

2.39 2.97 2.68

2.55

0.34 0.36 0.26

TABLE II - CONCENTRATIONS OF PROGESTERONE (ng/ml) IN SERUM AND MILK TAKEN ON THE DAY OF BREEDING AND 21 DAYS LATER. SERUM PROGESTERONE WAS ASSAYED BY THE AQUEOUS ASSAY AND MILK PROGESTERONE WAS ASSAYED BY THE SOLID-PHASE ASSAY. DIAGNOSES WERF. MADE ACCORDING TO THE CONCENTRATION OF PROGESTERONE AND VERIFIED BY RECTAL PALPATION

COW

number

711 766 875 710 740 383 867 882 854 705 816 401 648

Diagnosis

NP P P P P P P P P P* NP P P

Milk progesterone Day 0 Day 21

Serum progesterone Day 0 Day 21

0.44 0.25 0.18 0.15 0.24 0.33 0.23 0.10 0.16 0.08 0.23 0.23 0.x

0.81 0.09 0.15 0.08 0.07 0.07 0.85 0.76 1.6 0.52 0.34 0.81 0.55

0.19 7.6 7.2 4.7 7.2 4.5 8.2 3.9 6.8 5.3 0.25 3.2 6.1

Not pregnant when examined per rectum approximately breeding.

DECEMBER

1979VOL.12N0.6

0.80 3.0 3.2 4.6 10.7 3.7 5.1 4.0 5.2 3.1 0.34 7.0 7.5

60 days after

337

'60

43

41

21 day only

23 day only

I

I45

36

23 day + 0 day

/ 56

'48

38

21 day + 0 day

(73.2)

(71.7)

(80.0)

(79.2)

Pregnant

19, 20

19, 19

17, 18

18, 18

(95.0)

(100 )

(94.4)

(100 )

Not Pregnant

7, 70

8, 74

-

-

(10.0)

(10.8)

Questionable

USING ONLY ORIGINAL VALUES

I

/ 20

19

20,

18

'19 18,

19

42/56

(75.0)

(72.9)

(79.6)

(78.4)

20

'49

51

(95.0)

(100 )

(100 )

(100 )

Not Pregnant

43/ 59

39

40,

Pregnant

3,

4,

70

74

-

-

J

(4.3)

(5.4)

Questionable

USING ONLY REASSAYED VALUES

TABLE III - ACCURACY OF PREGNANCY DIAGNOSIS BY THE SOLID-PHASEASSAY USING VARIOUS COMBINATIONS OF VALUES IN MILK TAKEN ON THE DAY OF BREEDING (O), or 23 DAYS AFTER BREEDING. THE FINAL DIAGNOSIS WAS MAD BY RECTAL EXAMINATIONSPERFORMED 45 to 60 DAYS AFTER BREEDING. VALUES IN PAREN ??HESES ARG PERCENT CORRECT DIAGNOSES, THE NUMERATOR IS THF.NUMBER OF COWS DIAGNOSED CORRECTLY AND THE DENOMINATOR IS THE TOTAL NUMBER OF COWS. SEE TEXT FOR THE ASSIGNMENT OF LIMITS TO EACH CLASSIFICATION

THERIOGENOLOGY TABLE IV - MEAN, STANDARD DEVIATION AND RANGE OF CONCENTRATIONSOF PROGESTERONEIN PREGNANT AND NON-PREGNANTCLASSIFICATIONS

Classification

Not pregnant Itlea" s-d. range Pregnant mean s.d. range

DECEMBER

Sample taken on -Day.25 .30 .06-.97

Day 21 22 :21 .06-0.68

.20 .14 .06-.74

4.70 1.88 2.10-8.20

1979 VOL. 12 NO. 6

Day 23 .19 12 .06:.38

4.57 2.09 1.55-10.15

339

THERIOGENOLOGY

80-

‘//od5

Fig. 1.

340

.05 50 Progesterone hg /tube)

, 50

Standard curves for the solid-phase assay using 0.8 ml of gelatinphosphate buffer and (a) 0.11111 of 1.25% casein buffer (o-------o), (b) 0.1 ml of milk from which endogenous progesterone was removed by charcoal (+-+), (c) 0.1 ml of milk from an overiectomized cow (.-----.) and (d) 0.1 ml of gelatin-phosphate buffer (G-0). The binding of 1251-progesterone (B) at the various concentrations of unlabelled progesterone was expressed as a percent of the 0 standard (B,).

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY

F 3

30-

$ 28c” 2 CI 26‘;i i% 2 24-

a I

” -0 0‘

22-

I *.

‘4

I

0

2

4

6

8 Hours

Fig.

2.

10 of

12

14

1979 VOL. 12 NO. 6

I

18-

Incubation

125 The binding of I-progesterone to antibody-coated the absence of unlabelled hormone at 37’C cm), for 1 to 18h. (o---o) and 4’C (i- ---+)

DECEMBER

16

tubes in 22’=C

341

THERIOGENOLOGY

100 go-i\

80-

70-

60l! 507 2 b40g g30-

ZO-

10-

0’

8

Fig. 3.

342

24

16 Hours

of

32

40

48

56

Sedementation

Relative concentration of fat in milk after 12, 24 or 50 hours of sedimentation. Samples from 20 cows were allowed to separate and the ratio of surface fat:total milk was measured in cm Samples from 10 cows were allowed to separate and C-j. the residual fat was measured by the IRMA technique (c--4).

DECEMBER

1979 VOL. 12 NO. 6

THERIOGENOLOGY

7-

I ! I I

6n=134 3 2 L

5-

2

A-

0” ‘i;

1

n=41

n 1158

! I I l I

3-

i

0

---I

0.062

0.5

1.0

1.5

P rogcstcrone

Fig. 4.

DECEMBER

2.0

2.5

16.0

( ng / ml m i I k )

Histogram of concentrations of progesterone in milk samples from cows in the pregnancy diagnosis trial, where "a" refers to the number of progesterone determinations within each group.

1979VOL.12NO.6

343