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Clinical Application of the Progesterone-in-Milk Test
Br. vet. J. (1976), 132, 538
CLINICAL APPLICATION OF THE PROGESTERONE-IN-MILK TEST BY H.
DonsoN AND
R. J.
FITZPATRICK
Department of Veterinary Clinical Studies, University of Liverpool, Leahurst, Neston, Wirral, Merseyside, L64 7 TE INTRODUCTION
A low concentration of progesterone in milk is an indication that the animal, from which the milk was obtained, is not pregnant. It is the non-pregnant cow which provides the theme of the present paper and, basically, two main aspects will be considered: a very small, but intensive, investigation was carried out using the concentration of progesterone in milk to distinguish quickly those failing to conceive; and the progesterone content of milk from sub-fertile cows was determined to aid the choice of therapy. MATERIALS AND METHODS
The method used for progesterone measurement was the same for both studies. Based on that of Heap, Gwyn, Laing & Walters (I973), the modifications have already been described in detail elsewhere (Dobson, Midmer & Fitzpatrick, I975)· In summary, 20 ml milk was collected and stored at 4°C with added preservative until assayed. An aliquot of milk 25 [Ll was incubated directly with antiserum (raised against I I ex. hydroxy-progesterone-succinyl-BSA) and 3 H-progesterone. Separation of antibody-bound and unbound progesterone was achieved with a charcoal suspension. The results were compared to a standard curve prepared initially from known amounts of progesterone in 25 {Ll milk of low progesterone content. The majority of samples were obtained from one herd for the investigation into the practicalities of a pregnancy test based on the concentration of progesterone in milk. In order to reduce interruption of the milking routine, samples were ta ken by the cowman twice a week on Mondays and Thursdays, as near to 2 I days after insemination as possible, hence the range was 19 to 23 days after A.I. Earlier results obtained from plasma analyses (Dobson, Hopkinson & Ward, 1973), had suggested that this would be the most suitable sampling period. Once a week progesterone content was assessed and the results reported to the farmer. Seven to eight weeks after insemination, the cows were examined per rectum for pregnancy by experienced veterinarians. The main problem with the chemical test was to decide the exact end point upon which to base the diagnosis. It was accepted that the end point would
CLINICAL APPLICATIONS
539
vary between laboratories: a concentration ofless than 5 ·o ng progesteronefml milk was chosen to indicate unsuccessful insemination, and above 7·0 ngfml to indicate conception; samples between 5·0 and 7·0 ngfml were reported as doubtful. RESULTS
Over a period of nine months, 289 samples were analysed from 22 I cows: unsuccessful inseminations provided more than one sample from some cows. Seventysix cows (26%) were diagnosed non-pregnant with milk progesterone concentrations of less than 5·0 ngfml; I5 (5%) were in the doubtful category, and rg8 (6g% ) were diagnosed as pregnant. More detailed analysis of each group showed that of the 76 non-pregnant cows 6g (gi %) were subsequently confirmed non-pregnant by examination per rectum. In the milk test these cows had a mean concentration 2·1 ngfml (range s-o to I·o ng/ml). The remaining seven cows (g%) which had been diagnosed non-pregnant were found to be pregnant by examination per rectum; in the milk test they had a mean value of 3·5 ng/ml (range 4·5 to I ·o ngfml) i.e. 19 to 23 days after A.I. they were TABLE I SUMMARY OF DIAGNOSTIC TEST BASED ON MILK PROGESTERONE CONTENT
Diagnosis (n) Non-pregnant (76) Doubtful ( 15) Pregnant ( 198)
%
correct
Mean progesterone concentration
%
incorrect
Mean progesterone concentration
91
2·1 ngfml
9
3·5 ng/ml
81
15"7 ngfml
19
9·0 ng/ml
indistinguishable from the correctly diagnosed animals. In the doubtful category, 12 cows (8o%) were proved pregnant by examination per rectum; mean progesterone 5"38 ngfml (range 7"0 to 5"0 ngfml). Three COWS in this doubtful group were non-pregnant seven to eight weeks after A. I; mean progesterone at 2I days was 5·7 ngjml (range s·5 to 6·o ngfml). In the group of cows diagnosed pregnant, I6r (8I %) were subsequently proved to be correct (mean progesterone I5"7 ngjml: range 20 to 8 ngfml), but 37 (Ig%) were subsequently found non-pregnant. Examination of the records revealed that I3 animals (7% of the total pregnant group) were sampled at the wrong time, outside I9 to 23 days after A.l. Seven (4%) subsequently became clinical cases, mainly with luteal cysts, but for I 7 (g%) there was no explanation, mean progesterone g·o ng/ml (range 20 to 7 ngfml). A summary of these results is given in Table I. · DISCUSSION
The main conclusion drawn from this investigation was that the progesterone content of milk gave a more reliable indication that a cow was non-pregnant
BRITISH VETERINARY JOURNAL, 132, 5
540
rather than pregnant. Only 9% of the non-pregnant diagnoses were incorrect, compared to 19% incorrect pregnant predictions. The latter group consisted of cows which had high milk progesterone concentrations due to causes other than pregnancy : incorrect timing of sampling and prolonged luteal function were two which were defined in the present study. When considering the further development of a test to detect conception, it may be profitable to bear this in mind and measure substance (s) more specific to pregnancy e.g. originating from the conceptus. v'fhe progesterone content of milk in the non-pregnant cow has been considered m a clinical sub-fertility study carried out on several farms. 16
12
E
'coc:
.; c:
0
~.,
8
co 0
ct 4
Days · before and after oestrus
Fig. 1 . Mean concentration of progesterone (ng/ml) in milk ( A - - - - A ) and plasma ( e - -• ) from four cows during the oes trous cycle.
The animals were classed as infertile due to causes other than anatomical defects, disease or nutntwn: it was presumed the sub-fertility was due to an endocrine imbalance. The results from this survey have provided interesting information but are not considered here: it is the application of the progesterone milk test which is of importance in the present context. As the sub-fertility study had begun by establishing hormonal profiles in plasma, it was essential' to prove that changes in milk content paralleled those in plasma. Consequently, daily samples of plasma and milk were taken simultaneously from four cows, and progesterone results are shown in Fig. 1. The plasma concentrations were similar to those already reported (Dobson et al., 1973). The milk values confirmed those of Heap et al. (1973), being greater than plasma values. In addition, this study
CLINICAL APPLICATIONS
54 I
showed a very close correlation (regression coefficient r = o·88) between milk and plasma progesterone concentrations, suggesting that it would be usefuL to follow milk progesterone values in order to monitor endocrine changes in clinical cases both before and after treatment. All the milk samples so far considered have been taken from individual collecting jars at the completion of milking for each cow. In practical terms it would be advantageous for the veterinarian to be able to collect a milk sample upon clinical examination of an infertile cow. Therefore the progesterone content of milk taken by hand from each quarter after stripping the teat was compared to that obtained from the collecting jar immediately after. The results from six cows are shown in Table II; for each cow the values obtained from individual teats were very similar. There was only one sample from the collecting jar which varied greatly from the results from individual teats (cow 43). Thus, it was considered that this method of obtaining a milk sample provided an adequate assessment of the progesterone content of milk, thereby an indication of luteal function and, hence, which hormonal therapy would be TABLE II COMPARISON OF PROGESTERONE CONCENTRATION FROM DIFFERENT SOURCES
Progesterone concentration (ngfml) in milk Cow No.
From each teat
w·o,
32 36
I I·o, Ig·o, Io·o 3 '0, g·o, 3·5, 2'0 I·O, I·5, I·5, 1'5 2·0, I·5, o·s, 2'0 12·0, 10·0, B·o, I2'0
43
B·o, B·o, g·o, w ·o
2
3 6
From collecting jar I'5 2'0 4'0 12'0 22'0
most efficacious. However, as a diagnostic test for the veterinarian in practice, it is doubtful whether the information would be of great value owing to the delay required for assessment of progesterone concentrations. In many situations, the endocrine balance changes hourly, so that the information is needed immediately to be of real use. The future and application of the milk progesterone test considered in the light of the foregoing work lies in further development. As a method of pregnancy diagnosis, the test can only be reliable to determine non-pregnancy and its efficiency for this must be considered very carefully. Esslemont ( 1975) and Morris (1973) have shown that great economical savings can be made by increased vigilance in observing oestrous behaviour (another positive indication of non-pregnancy). Such a method would be cheaper and as effective as the present radioimmunoassays for progesterone, and immediate recognition of non-pregnancy by observing oestrus could be followed by direct action to bring about insemination. Greater significance may be attached to a new test which is specific to pregnancy and emphasis should be laid on attempts to develop such a method. Restrictions on timing of the test would not be so rigid
542
BRITISH VETERINARY JOURNAL, 132,- 5
and it could be repeated later in pregnancy. However, as with the pregnant mare's serum gonadotrophin test for horses, errors can arise immediately after embryonic death when the placental hormone is still present in plasma. For use as an adjunct to clinical diagnosis, the milk progesterone test could be useful, although development of a method which can be carried out on the farm, without recourse to a laboratory, is really necessary. ACKNOWLEDGEMENTS
Gratitude is expressed to Messrs J. E. F. Rankin and W. R. Ward for veterinary expertise; to Sandie Mid mer for technical assistance; to Messrs. T. A. and R. N. Dobson and to J. Bibby and Company for access to their dairy herds; to the Animal Health Trust for a Wooldridge Fellowship to Hilary Dobson; to the Agricultural Research Council for financial support; to the Milk Marketing Board for milk preservative tablets; to the Editor of the Veterinary Record for permission to reprint Fig. I. REFERENCES
DoBSON, H ., HoPKINSON, C. R . N. & WARD, W. R . ( 1973). Vet . Rec. 93, 76. DoBSON, H ., MIDMER, S. E . & FITZPATRICK, R. J. (1975). Vet . Rec. 9«i, 222 . EssLEMONT, R. J . (1975). Vet. A. 15th ed . p. 50. HEAP, R. B., GwYN, M., LAING,]. A . & WALTERS, D. E. (1973). ]. agric. Sci., Camb. 8r, 151. MORRIS, R . S. (1973 ). Vet. &c. 92, 16.
CLINICAL APPLICATION OF THE PROGESTERONE-IN-MILK TEST BY H.
DonsoN AND
R. J.
FITZPATRICK
Department of Veterinary Clinical Studies, University of Liverpool, Leahurst, Neston, Wirral, Merseyside, L64 7 TE INTRODUCTION
A low concentration of progesterone in milk is an indication that the animal, from which the milk was obtained, is not pregnant. It is the non-pregnant cow which provides the theme of the present paper and, basically, two main aspects will be considered: a very small, but intensive, investigation was carried out using the concentration of progesterone in milk to distinguish quickly those failing to conceive; and the progesterone content of milk from sub-fertile cows was determined to aid the choice of therapy. MATERIALS AND METHODS
The method used for progesterone measurement was the same for both studies. Based on that of Heap, Gwyn, Laing & Walters (I973), the modifications have already been described in detail elsewhere (Dobson, Midmer & Fitzpatrick, I975)· In summary, 20 ml milk was collected and stored at 4°C with added preservative until assayed. An aliquot of milk 25 [Ll was incubated directly with antiserum (raised against I I ex. hydroxy-progesterone-succinyl-BSA) and 3 H-progesterone. Separation of antibody-bound and unbound progesterone was achieved with a charcoal suspension. The results were compared to a standard curve prepared initially from known amounts of progesterone in 25 {Ll milk of low progesterone content. The majority of samples were obtained from one herd for the investigation into the practicalities of a pregnancy test based on the concentration of progesterone in milk. In order to reduce interruption of the milking routine, samples were ta ken by the cowman twice a week on Mondays and Thursdays, as near to 2 I days after insemination as possible, hence the range was 19 to 23 days after A.I. Earlier results obtained from plasma analyses (Dobson, Hopkinson & Ward, 1973), had suggested that this would be the most suitable sampling period. Once a week progesterone content was assessed and the results reported to the farmer. Seven to eight weeks after insemination, the cows were examined per rectum for pregnancy by experienced veterinarians. The main problem with the chemical test was to decide the exact end point upon which to base the diagnosis. It was accepted that the end point would
CLINICAL APPLICATIONS
539
vary between laboratories: a concentration ofless than 5 ·o ng progesteronefml milk was chosen to indicate unsuccessful insemination, and above 7·0 ngfml to indicate conception; samples between 5·0 and 7·0 ngfml were reported as doubtful. RESULTS
Over a period of nine months, 289 samples were analysed from 22 I cows: unsuccessful inseminations provided more than one sample from some cows. Seventysix cows (26%) were diagnosed non-pregnant with milk progesterone concentrations of less than 5·0 ngfml; I5 (5%) were in the doubtful category, and rg8 (6g% ) were diagnosed as pregnant. More detailed analysis of each group showed that of the 76 non-pregnant cows 6g (gi %) were subsequently confirmed non-pregnant by examination per rectum. In the milk test these cows had a mean concentration 2·1 ngfml (range s-o to I·o ng/ml). The remaining seven cows (g%) which had been diagnosed non-pregnant were found to be pregnant by examination per rectum; in the milk test they had a mean value of 3·5 ng/ml (range 4·5 to I ·o ngfml) i.e. 19 to 23 days after A.I. they were TABLE I SUMMARY OF DIAGNOSTIC TEST BASED ON MILK PROGESTERONE CONTENT
Diagnosis (n) Non-pregnant (76) Doubtful ( 15) Pregnant ( 198)
%
correct
Mean progesterone concentration
%
incorrect
Mean progesterone concentration
91
2·1 ngfml
9
3·5 ng/ml
81
15"7 ngfml
19
9·0 ng/ml
indistinguishable from the correctly diagnosed animals. In the doubtful category, 12 cows (8o%) were proved pregnant by examination per rectum; mean progesterone 5"38 ngfml (range 7"0 to 5"0 ngfml). Three COWS in this doubtful group were non-pregnant seven to eight weeks after A. I; mean progesterone at 2I days was 5·7 ngjml (range s·5 to 6·o ngfml). In the group of cows diagnosed pregnant, I6r (8I %) were subsequently proved to be correct (mean progesterone I5"7 ngjml: range 20 to 8 ngfml), but 37 (Ig%) were subsequently found non-pregnant. Examination of the records revealed that I3 animals (7% of the total pregnant group) were sampled at the wrong time, outside I9 to 23 days after A.l. Seven (4%) subsequently became clinical cases, mainly with luteal cysts, but for I 7 (g%) there was no explanation, mean progesterone g·o ng/ml (range 20 to 7 ngfml). A summary of these results is given in Table I. · DISCUSSION
The main conclusion drawn from this investigation was that the progesterone content of milk gave a more reliable indication that a cow was non-pregnant
BRITISH VETERINARY JOURNAL, 132, 5
540
rather than pregnant. Only 9% of the non-pregnant diagnoses were incorrect, compared to 19% incorrect pregnant predictions. The latter group consisted of cows which had high milk progesterone concentrations due to causes other than pregnancy : incorrect timing of sampling and prolonged luteal function were two which were defined in the present study. When considering the further development of a test to detect conception, it may be profitable to bear this in mind and measure substance (s) more specific to pregnancy e.g. originating from the conceptus. v'fhe progesterone content of milk in the non-pregnant cow has been considered m a clinical sub-fertility study carried out on several farms. 16
12
E
'coc:
.; c:
0
~.,
8
co 0
ct 4
Days · before and after oestrus
Fig. 1 . Mean concentration of progesterone (ng/ml) in milk ( A - - - - A ) and plasma ( e - -• ) from four cows during the oes trous cycle.
The animals were classed as infertile due to causes other than anatomical defects, disease or nutntwn: it was presumed the sub-fertility was due to an endocrine imbalance. The results from this survey have provided interesting information but are not considered here: it is the application of the progesterone milk test which is of importance in the present context. As the sub-fertility study had begun by establishing hormonal profiles in plasma, it was essential' to prove that changes in milk content paralleled those in plasma. Consequently, daily samples of plasma and milk were taken simultaneously from four cows, and progesterone results are shown in Fig. 1. The plasma concentrations were similar to those already reported (Dobson et al., 1973). The milk values confirmed those of Heap et al. (1973), being greater than plasma values. In addition, this study
CLINICAL APPLICATIONS
54 I
showed a very close correlation (regression coefficient r = o·88) between milk and plasma progesterone concentrations, suggesting that it would be usefuL to follow milk progesterone values in order to monitor endocrine changes in clinical cases both before and after treatment. All the milk samples so far considered have been taken from individual collecting jars at the completion of milking for each cow. In practical terms it would be advantageous for the veterinarian to be able to collect a milk sample upon clinical examination of an infertile cow. Therefore the progesterone content of milk taken by hand from each quarter after stripping the teat was compared to that obtained from the collecting jar immediately after. The results from six cows are shown in Table II; for each cow the values obtained from individual teats were very similar. There was only one sample from the collecting jar which varied greatly from the results from individual teats (cow 43). Thus, it was considered that this method of obtaining a milk sample provided an adequate assessment of the progesterone content of milk, thereby an indication of luteal function and, hence, which hormonal therapy would be TABLE II COMPARISON OF PROGESTERONE CONCENTRATION FROM DIFFERENT SOURCES
Progesterone concentration (ngfml) in milk Cow No.
From each teat
w·o,
32 36
I I·o, Ig·o, Io·o 3 '0, g·o, 3·5, 2'0 I·O, I·5, I·5, 1'5 2·0, I·5, o·s, 2'0 12·0, 10·0, B·o, I2'0
43
B·o, B·o, g·o, w ·o
2
3 6
From collecting jar I'5 2'0 4'0 12'0 22'0
most efficacious. However, as a diagnostic test for the veterinarian in practice, it is doubtful whether the information would be of great value owing to the delay required for assessment of progesterone concentrations. In many situations, the endocrine balance changes hourly, so that the information is needed immediately to be of real use. The future and application of the milk progesterone test considered in the light of the foregoing work lies in further development. As a method of pregnancy diagnosis, the test can only be reliable to determine non-pregnancy and its efficiency for this must be considered very carefully. Esslemont ( 1975) and Morris (1973) have shown that great economical savings can be made by increased vigilance in observing oestrous behaviour (another positive indication of non-pregnancy). Such a method would be cheaper and as effective as the present radioimmunoassays for progesterone, and immediate recognition of non-pregnancy by observing oestrus could be followed by direct action to bring about insemination. Greater significance may be attached to a new test which is specific to pregnancy and emphasis should be laid on attempts to develop such a method. Restrictions on timing of the test would not be so rigid
542
BRITISH VETERINARY JOURNAL, 132,- 5
and it could be repeated later in pregnancy. However, as with the pregnant mare's serum gonadotrophin test for horses, errors can arise immediately after embryonic death when the placental hormone is still present in plasma. For use as an adjunct to clinical diagnosis, the milk progesterone test could be useful, although development of a method which can be carried out on the farm, without recourse to a laboratory, is really necessary. ACKNOWLEDGEMENTS
Gratitude is expressed to Messrs J. E. F. Rankin and W. R. Ward for veterinary expertise; to Sandie Mid mer for technical assistance; to Messrs. T. A. and R. N. Dobson and to J. Bibby and Company for access to their dairy herds; to the Animal Health Trust for a Wooldridge Fellowship to Hilary Dobson; to the Agricultural Research Council for financial support; to the Milk Marketing Board for milk preservative tablets; to the Editor of the Veterinary Record for permission to reprint Fig. I. REFERENCES
DoBSON, H ., HoPKINSON, C. R . N. & WARD, W. R . ( 1973). Vet . Rec. 93, 76. DoBSON, H ., MIDMER, S. E . & FITZPATRICK, R. J. (1975). Vet . Rec. 9«i, 222 . EssLEMONT, R. J . (1975). Vet. A. 15th ed . p. 50. HEAP, R. B., GwYN, M., LAING,]. A . & WALTERS, D. E. (1973). ]. agric. Sci., Camb. 8r, 151. MORRIS, R . S. (1973 ). Vet. &c. 92, 16.