Use of the Milk Progesterone Test for Pregnancy Determination

Br. vet. j. (\9 79 ), 135,478

/ USE OF THE MILK PROGESTERONE TEST FOR PREGNANCY DETERMINATION \ By]. M. BOOTH, ]' DAVIES AND R.]. HOLDSWORTH

Miik Marketing Board Veterinary Laboratory, Cieeve House, Lower Wick, Worcester WR2 4NS

SUMMARY

A pregnancy testing service using the milk progesterone test has been available to dairy farmers in the United Kingdom since 1975. After three years operation, more than 100000 cows a year are being tested and 5·6% of the farmers in England and Wales are using the service. The service is used proportionately more in larger herds (median herd size is 76 cows) and in autumn-calving herds. The percentage of negative tests rose to 20·0% in the third year, although there was considerable seasonal variation. A sample of large herds using the service found average accuracy rates of 84·5% and 97·0% respectively for positive and negative tests. A highly significant correlation (r= + 0·84) was found between the proportion of positive tests in a herd and the accuracy rate of these tests. These two factors, and analysis of insemination interval data, are good indicators of herds requiring veterinary investigation. INTRODUCTION

The use of milk progesterone measurement for the determination of pregnancy was first suggested by Laing & Heap (1971), and subsequent work by Heap and co-workers (Heap et ai., 1973; Heap et ai., 1976) demonstrated the use of a direct rapid radioimmunoassay for pregnancy diagnosis. Other workers have used similar techniques to determine the concentration of progesterone in milk for this and related veterinary clinical and research purposes (Hoffman & Hamburger, 1973; Dobson, Midmer & Fitzpatrick, 1975; Pope & Hodgson-Jones, 1975; Schiavo et ai., 1975; Hoffman et ai., 1976; Laing, 1976; Lamming & Bulman, 1976; Pennington, Spahr & Lodge, 1976a). Booth & Holdsworth (I976) described the organization of a laboratory to receive and test large numbers of milk samples for pregnancy determination. The current report details progress in the application of the test in the field. As described previously, samples of whole milk are taken by the farmer from the afternoon milking 24 days after insemination, preserved and posted to the testing laboratory. The radioimmunoassay technique is based closely on the method described by Heap et al. (1976), wIth modifications as described by Holdsworth, Chaplin & Booth (1979). Milk progesterone levels below 5·5 ng/ml are reported as negative, i.e. not

479

MILK-PROGESTERONE PREGNANCY TEST

pregnant, above 7·5 ng/ml they are reported as positive, i.e. pregnant, and repeat samples at 42 days after insemination are requested from approximately 2% of cows which fall between these levels and which are reported as doubtful. In order to maintain the essence of an early pregnancy testing service, reports on the majority of sampks are despatched to the farmer within two working days of receipt.

RESULTS

At the end of the third year of operation 2842 farmers were using the pregnancy testing service. They represented 5·6% of the 48 000 dairy farmers in England and Wales (Table I) with a small number of members in Scotland, Northern Ireland, the Isle of Man and the Channel Islands. Table I illustrates the very variable regional uptake, with approximately one in six of the farmers in South East England using the service compared to only one in 40 in Wales. TABLEI MEMBERSHIP OF PREGNANCY TESTING SERVICE, SEPTEMBER 197 8

Members Dairy farmers

No.

South Eastern Eastern Southern East Midland Wes~ Midland MidWestern Northern North Western Far Western South Wales North Wales

1841 1316 1652 2000 4260 5317 5180 10945 6839 5785 2982

298 168 202 211 281 337 223 471 292 161 68

16·2 12 ·8 12 ·2 10·6 6·6 6·3 4·3 4·3 4·3 2·8 2·3

England and Wales

48 117

2712

5·6

Region

%

The milk pregnancy test has appealed particularly to the owners oflarger herds. The current median herd size of members is 76 cows, although herd sizes vary from two t? over 500 cows; the average herd size in England and Wales is 50 cows (U.K. Dairy Facts & Figures, 1978). This selective uptake appears to go some way to explaining the variable regional pattern of membership. There is a strong correlation (r=+0·87) between the regional membership, on a percentage basis, and the average herd size of the region (Fig. 1). On this calculation, as the herd size of a region increases by 10 cows so membership of the service could be expected to increase by 2·6% of dairy farmers in that region. The relationship is not as simple as this however because, apart from a trend towards increasing herd sizes, approximately 2500 dairy farmers go out of milk production each year in England and Wales.

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The number of cows tested annually is now over 100000 (Table II ) and is continuing to rise. From the start of the service in October 1975, there has been a marked seasonality in sample receipt. Fig. 2 demonstrates that over 50% of samples in the third year were received in the four-month period January to April, and this pattern is becoming marginally more pronounced each year. It had been expected (Booth & Holdsworth, 1976) that the receipt of milk samples would relate closely to the artificial insemination figures for the country, following approximately one month behind the peaks and troughs. This has never been the case however and Fig. 3 demonstrates that growth has consistently been during the first few months of the year. Interestingly there has been a tendency towards the concentration of inseminations during the November to January period (Fig. 4), which is roughly in in line with the change in sample receipt observed above. Our earlier .report commented on the very high proportion of positive milk tests, 88 ·5% at that time, and the considerable difference from the average AI non-return TABLE II NUMBER OF

cows TESTED

AND RES ULTS OF TESTS

Year

No . cows tested

Positive (%)

Negative (%)

Doubtful (%)

197 5/76 1976/7 7 1977/78

58286 91888 107518

84 ·4 81·4 77 ·9

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rate of approximately 79%. Since that time there has been a progressive decline in the proportion of positive tests (Table II ) with a corresponding increase in negative tests; there has been no significant change in assay characteristics or discriminatory limits during this period. The proportion of negative tests demonstrates a consistent seasonal pattern (Fig. 5), with fewer non-pregnant cows in the period October to January, and more during March to May and again in September. Examination of the relationship between pregnancy test results and the AI 30/~0 day non-return rate indicated a significant correlation during 1976/7 7, but this no longer holds true. In separate investigations (Booth, in preparation), we have found somewhat higher milk progesterone levels in Jersey cows (28·6±0·8 ng/mll than in Friesian cows (25·3 ±0 ·7 ng/ml). In common with Pennington, Spahr & Lodge (l976b) however, we have found that this small difference does not in practice affect the accuracy of the test. Wider investigations have indicated that within-breed variations of milk progesterone levels are very much greater than between-breed variations. Accuracy of the milk progesterone test for pregnancy has been continuously monitored, both by means of field trials and by surveys of members of the service. In recent field trials in four herds, a mean accuracy rate of 85 ·2% has been found for positive milk tests (Booth, in preparation). Individual herd accuracy rates however varied greatly, from 100% down to 67 ·4%, and this has also been a consistent finding in

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surveys. Accuracy of negative tests in these field trials was 100% in all but one herd . From a sample of 30 herds where the milk pregnancy test had been used on more than 100 cows in 1976/7 7, full return to service or veterinary rectal examination data were received from 10 herds with a total of over 2400 cows. The overall distribution of test results for these herds (Table III) was similar to the national situation. Individual herds however varied considerably; the proportion of negative tests varied from 7 ·5% TABLE III TEST RESU L TS I N TEN SURVEY HERDS (I 9 76/77 )

Herr! 53 S5 59 S11 S12 S13 S14 S15 S16 S17 Overall means

Total cows tested

Positive

Test results Negative

%

%

%

139 173 329 287 334 190 133 227 339 278

79 ·9 90 ·2 78· 7 83·6 64 · 7 77 ·4 84·2 85 ·0 67·0 78 ·8

18 · 7 7·5 18·2 15·3 32·0 21 ·6 12 ·8 13·2 31 ·8 18 · 7

2·3 3·1 1· 1 3·3 1·0 3·0 1·8 1·2 2·5

19 ·9 21·0 20 ·2 21 · 7 19 ·3 20·6 24·3 22·0 21·0 24·0

(2429)

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Mean progesterone ofpositives (nglml)

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Fig. 6. Accuracy o rtests in survey herds, 197617 7.

to 32·0% for example. The mean milk progesterone values of the positive tests varied comparatively little from herd to herd, ranging from 19·3 ng/ml to 24 ·3 ng/ml; this is in agreement with previous findings (Booth & Holdsworth, 1976). Fig. 6 illustrates the range of accuracy rates achieved in this group of herds . Overall the positive tests were 84 ·5% accurate and the negative tests 97 ·0%accurate. However, as shown in the figure, the range for positive test results was very wide indeed, varying from 98 ·2% (Herd S14) down to 72 ·2% (Herd SI2); there is some clustering in the range 85 to 90%. Interestingly, there was a highly significant relationship (r= + 0·84 ) between the proportion of positive test results in a herd and the accuracy rate of these results (Fig. 7); for a decline of 10% in positive test results there was a reduction in accuracy rate of the positive tests of8%. Accuracy of the negative test results was high, with four of the 10 herds having a 100% accuracy rate and all being over 93% (Fig. 6). There was a total of 15 false negative tests (3%) . Eight of these had appreciable milk progesterone levels, several being close to the discriminatory limit. Three incorrect test results in one herd were all contained in one assay which subsequently came under suspicion for other reasons, and a further cow calved after a gestation period indicative of a subsequent insemina· tion. The remaining three false negative tests could not be explained, although there was the suggestion of incorrect cow identification in at least one instance. The data received from field trial and survey herds have been analysed according to insemination intervals, using tfie mtervals ot Hoyd & Reed (I 961 ). Considerable between-herd variation has been revealed (Table I"V ). Herds SIB and R33 are fairly typical survey herds, with reasonable oestrus detection, although both have sutfered it

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TABLEIV I NSEMI NATIO N INTERVALS

Percentage oj returns to service SiB

R33

Herd no. RiA

RiB

RiC

Boyd & Reed (196i )

(1973)

< 18 18-24 25-35 36-48 > 48

4·9 46 ·5 18 · 7 16 · 7 13·2

6· 7 47 · 1 18 ·3 17 ·3 10·6

2·9 18 ·3 19 ·2 26 ·9 32 ·7

3·0 31 ·3 25· 4 20 ·9 19 ·4

0 18 ·4 22 ·4 20 ·4 38·8

3· 0 47 ·8 10·2 17 · 1 2 1·9

5· 4 43 ·5 7·6 16 ·9 26 ·6

Total returns to service

144

104

208

67

98

1709

184

Interval (days)

Boyd

degree of infertility as evidenced by comparatively high numbers of negative milk tests at certain times of the year. Both herds have approximately 47% of insemination intervals falling into the period of 18 to 24 days after a previous insemination. This is in close agreement with data derived from the work of Boyd & Reed (1961) and Boyd (197 3) which has been included for comparison. Field trial herds RIA, RIB and RIC appear abnormal by these criteria, with even the best one only having 31% of insemination intervals falling in the 18 to 24-day period ; a contributory factor to this situation is believed to be the fact that all three include valuable pedigree cows. Herd RIC has a known foetal mortality problem, which may explain the high proportion of cows returning more than 35 days after insemination ; almost 60% compared to the

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BRITISH VETERINARY JOURNAL, 135, 5

normal 30 to 40% range. Oestrus detection is known to be poor in herd RIA, which probably accounts for the high proportion of longer intervals in this herd.

DISCUSSION

The milk progesterone test for pregnancy plays a valuable role for the modern dairy farmer with a large herd and a tight calving pattern, where the optimum calving interval can have a pronounced financial impact. Approximately 5% of the dairy cows in England and Wales are currently tested, and the service is continuing to expand. Large herds are more Jikejy to use the milk pregnancy.,.test and the 16% uptake in South-Eastern England indicates the probability of further expansion. In addition, substantially more use of the service is made in autumn-calving herds than in spring/summer or all-the-year-round calving herds so that, with recent AI figures indicating a still greater concentration of calving in the autumn, a higher uptake of milk pregnancy testing may be expected. Esslemont & Eddy (1977) have quoted an improvement in milk production of 2· 7 gallons (12·3 litres) per cow for each day that the calving interval is reduced down to 365 days. At 1978 prices, and after allowing for feed costs, this represents a benefit of 83 pence per cow per day. For one missed oestrous cycle the loss is thus over £ 17. At the current cost of the service (75 pence + Value Added Tax of 8%) the value of the milk progesterone test is clearly demonstrated. The pronounced and consistent seasonal variation in milk test results had not been anticipated although non-return rates to AI had indicated that there might be some variation. However, after it appeared that there was a relationship between test results and AI non-return rates in the first months of the service, the correlation coefficient for the third year has now declined to virtually zero. In addition, the proportion of negative test results has risen by almost 50% over the three years of the service. With regard to the seasonal variation, one may postulate that a higher proportion of young cows are being tested in the early part of the season, O ctober to January, and that conception rates could be expected to be higher in these cows. Subsequently, older cows will be tested in March to May, at the same time as the refractory breeders from the early period are still being tested, resulting in a higher proportion of negative pregnancy tests. Once the cows are out at grass fertility may be expected to improve, producing a lower proportion of negative tests, with again a comparatively large proportion of the most difficult breeders being included in the small number of cows tested in September. Possible reasons for the year-to-year increase in negative tests are more dithcult to deduce. AI non-return rates for the three years are all between 79 and 80% with only a 0·5% decline in the third year. Other suggestions have included an increased awareness by farmers of the proportion of pregnant cows presented for insemination, which leads them to sample all cows including those apparently showing oestrus around 21 days after insemination, the use of the service to give difficult breeders additional opportunities to conceive, and even possible reliance on the milk pregnancy test to replace close observation for oestrus behaviour. The accuracy of the milk progesterone test is now fairly well established and most workers in the field have found accuracy rates around 85% for positive tests and close

MILK-PROGESTERONE PREGNANCY TEST

487

to 100% for negative tests. In small trials and surveys, where identification of animals should be totally reliable, higher positive accuracy rates have been produced. It is not strictly accurate to include foetal loss in the false positive category, as the cow would have been pregnant on the day of sampling, but in the farmer's eyes the milk test gave in inaccurate result and so, with reservations, this must be accepted . The reservations however may lead to additional use of the information already produced by the milk pregnancy test. In the 10 survey herds quoted earlier, the correlation between the proportion of positive test results and the accuracy rate of these positive results was highly significant. What this appears to mean, in this group of herds at least, is that in herds where conception is low there is also a higher than average level of foetal loss. In this small group of herds, there are two with a low proportion of positive tests, both below 70%, and a reduced accuracy rate of the positive tests, both below 80%. Such information could indicate herds with fertility problems requiring veterinary investigation. A number of farmers use the milk test for early detection of non-pregnant cows and the veterinary surgeon's examination at a later stage for confirmation of pregnancy, a course of action which the economics of the situation amply justify. It may well transpire that this test, by highlighting herds which suffer reduced fertility, will result in increased demand for the veterinary surgeon's professional expertise. One other indicator of herds requiring veterinary attention has been demonstrated by analysis of insemination interval data. A low proportion of returns to service in the 18 to 24-day period suggests poor oestrus detection, which would be confirmed by a higher than average number of negative milk tests, or a problem of foetal mortality, substantiated by higher than average false positive milk tests . The milk progesterone test for pregnancy determination can also be applied to species other than the bovine, for example the goat (Holdsworth & Davies, 1977; Pennington et at., 1977) and the mare (Bailes & Holdsworth, 1978; Hunt, Lein & Foote, 1978). The test has also been carried out in exotic species such as the bison and the yak. In the goat, extensive field trials have demonstrated accuracy rates of 86·8% and 100% for positive and negative milk tests respectively (Holdsworth & Davies, 1977) and a commercial service is now available. The technique is however unable to distinguish pseudo-pregnancy. The test seems unlikely to find application on a routine basis in other species with the possible exception of the sheep.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the advice and encouragement of Dr R. B. Heap and Mr G . F. Smith throughout this work, and the invaluable technical assistance of Miss V. M . Chaplin and Mrs P.] . Meadows.

'REFERENCES

BAILES, C. & HOLDSWORTH,R. j. (1978) . British Veterinary journal 134, 214. BOOTH,] . M. & HOLDSWORTH, R. j. (1976 ). British Veterinary journal 132, 518 . BOYD, H. (1973 ). Veterinary Record 92, 427 . BOYD, H . & REED, H . C . B. (1961). British Veterinaryjoumal1l7, 18 .

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DOBSON, H ., MIDMER, S. E. & FITZPATRICK, R . ]. (1975). Veterinary Record 96, 222 . ESSLEMONT, R . ]. & EDDY,R. G. (1977). British Veterinaryjoumal133, 346. HEAP, R . B., GWYN,M., LAING,]. A. &WALTERS,D . E . (1973).joumalofAgricultureScience, Cambridge 81,151. HEAP, R . B., HOLDSWORTH, R, j., GADSBY,]. E." LAING,]' A . & WALTERS, D . E., (1976). British . . , Veterinaryjoumal 132, 445. HOFFMANN, B . & HAMB URGER, R. (1973). Zuchthygiene 8, 154. HOFFMAN N, B., GUNZLER, 0. , HAMBURGER, R. & SCHMIDT, W . ( 1976). British Veterinaryjoumall32, 469 . HOLDSWORTH, R .j., CHAPLIN,V. M . &BOOTH,J. M . (1979 ). British Veterinaryjoumal, 135,454. HOLDSWORTH, R.]. & DAVIES,]. (1977). Abstracts of the Societyfor the Study ofFertility, Dec. 1977 . H UNT, B., LEIN, D. H. & FOOTE, R. H. (1978).joumaloftheAmerican Veterinary Medical Association 172, 1298. LAI NG,]' A . (1976). British Veterinaryjoumall32, 534. LAING,]' A. & HEAP, R . B . (1971). British Veterinaryjoumal127, xix. LAMMING, G. E . & B ULMAN, D . C . (1976). British VeterinaryJoumal132, 507. PEN NINGTON,]' A., SPAHR,S. L. & LODGE,]. R . (1976a).joumal ofDairy Science 59, 1528 . PENN IN GTON,]' A., SPAHR,S. L. & LODGE,]' R . (1976b). British Veterinaryjoumal132, 487. P ENNINGTON,]. A ., SPAHR, S. L., LODGE,]. R . & CONSIDINE,D . (1977).joumal of Dairy Science 60, Supplement 1,8 2. POPE, G. S . & HOD GSON] ONES,L. S. (1975). Veterinary Record 96, 154 . SCHIAVO,]., MATUSZCZAK, R. L., OLTENACU, E. B. & FOOTE, R. H. (1975).joumal of Dairy Science 58,1713 . U.K. DAIRY FACTS & FIGURES(l978). 35.