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Pregnancy testing of cows by determining progesterone levels in milk
Animal Reproduction Science, 4 (1981/1982) 181--187
181
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREGNANCY TESTING OF COWS BY DETERMINING LEVELS IN MILK
PROGESTERONE
J. Z A O R A L , M. P()SCHL* and J. KVAPILIK
Research Institute for Cattle Breeding, Rapotih 788 13 Vik~ovice (Czechoslovakia) *State Breeding Enterprises, General Directorate 252 O0 Hradi~tko pod Medn[kem (Czechoslovakia) (Accepted 30 June 1981)
ABSTRACT Zaoral, J., PSschl, M. and Kvapilfk, J., 1982. Pregnancy testing of cows by determining progesterone levels in milk. Anita. Reprod. Sci., 4: 181--187. In six herds, each comprising 400 to 600 cows, the progesterone test (PT) was compared at different numbers of milk samplings from one cow and at a reduced number of days of milk sampling per week (from seven to two per week). There were three variants. In four herds the foremilk was sampled on the 23rd day after insemination in the period from October 1978 to September 1979 (variant A). In the remaining two herds the samples were taken from the whole amount of milk from single milking in May to July 1979 (variant B) and in August to November 1979 (variant C). In variant B the samples were taken on the day of insemination or the first day after insemination, and on the 19th and 23rd days after insemination. In variant C also three samples were taken after each insemination, but only on two days in the week, Monday and Thursday ( l s t sampling 19--22, 2nd sampling 22--25 and 3rd sampling 29--32 days after insemination). A radioimmunological method was used to determine progesterone (tritium-labelled progesterone). Pregnancy was diagnosed by rectal palpation between the 60th and 90th days after insemination. In comparison with a single sampling, the use of two or three samples after insemination in variants B and C did not improve the proportion of correct results in the group of non-pregnant cows (progesterone level ~< 2.4 ng m1-1) nor in the overall results. Of the clinically identified non-pregnant cows, single milk sampling correctly determined 42.7 and 43.0%, the combination of two samplings 58.9 to 62.3%, and the combination o f three samplings 74.7% of the cows. The differences were significant (P < 0.01). The reduction of sampling days from seven to two in a week did not influence adversely the proportion of the results which were in agreement in the PT and clinical examination.
INTRODUCTION The direct radioimmunological determination of milk progesterone makes it possible to use this m e t h o d for d e t e r m i n i n g the success o f i n s e m i n a t i o n . T h i s is o f p a r t i c u l a r i m p o r t a n c e f o r l a r g e - c a p a c i t y c o w - h o u s e s in w h i c h t h e p r o c e s s o f r e p r o d u c t i o n is a r a t h e r d i f f i c u l t t a s k . I f t h i s m e t h o d is u s e d , t h e
0378-4320/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
182 service period is not unnecessarily prolonged and hence economic losses are prevented. The m e t h o d is particularly suitable for herds included in a controlled breeding programme (Wishart et al., 1975). An important prerequisite for a practical use of the progesterone test is the proportion of the results of this test which agree with the results of clinical examination. The majority of authors believe that non-pregnancy can be predicted with an accuracy of 95--100% and pregnancy with an accuracy of 75--85% (Hoffmann et al., 1974; Heap et al., 1976; Shemesh et al., 1978). Hence generally correct results can be expected in about 80% of the animals, and with two measurements (on the day of insemination and the 20th day after insemination) in 9 0 % of the animals. These results should be regarded as highly accurate and practically cannot be further improved (Gilnzler et al., 1975). Sampling on the day of insemination can eliminate inseminations performed at the wrong time. The proportion of such inseminations is 10--20% (Elsaesser, 1979). Paired sampling did n o t show a statistically significant improvement of the results in comparison with single samples (Heap et al., 1976). The combination of sampling on the 21st and 24th days after insemination is regarded as the best (the following sampling terms were used by Heap et al.: the 21st, 24th, 28th and 42nd days). In commercial service,rendered in Great Britain by the Milk Marketing Board, milk sampling is repeated on the 42r/~1 day if the results of sampling on the 24th day are dubious (Booth and Holdsworth, 1976). In this paper, single milk sampling is compared with double and/or triple samplings under farm conditions. The facilitation of samplings by reducing the number of sampling days per week is also discussed. MATERIAL AND METHODS A pilot experiment was conducted in six herds of 400 to 600 cows in each (mostly crossbreds between the Bohemian Red-Spotted and Holstein breeds). The experiment had three variants. Variant A -- in four herds foremilk was sampled on the 23rd day after insemination in the period from October 1978 to September 1979. In the remaining two herds milk was sampled from the whole a m o u n t of milk obtained in single milking. These two herds comprised variant B -- from May to July 1979 -- and variant C -- from August to November 1979. Variant B -- three samplings performed after each insemination. The first sample was taken on the day of insemination or one day later. The second sample was taken 19 days after insemination and the third sampling followed on the 23rd day after insemination. Variant C -- three samplings were performed after each insemination. The sampling days were two in a week (Monday and Thursday). The first sample was taken 19--22 days after insemination and the second was taken on Thursday if the first sampling had been performed on Monday and vice versa. The second sampling took place 22--25 days after insemination and the third
183 followed after 7 days, i.e. 29--32 days after insemination. A b o u t 2 ml milk was preserved with potassium dichromate and was kept in a refrigerator until dispatch or transport to the laboratory. The samples were sent by mail or transported by car to the laboratory once or twice a week. The radioimmUnological m e t h o d (RIA) with direct progesterone determination from whole milk after Pfchov~ et al. (1977) was used for the analysis of the milk samples. Milk (20 pl) was diluted with buffer (1 : 4) and then assayed. The specific a n t i b o d y used, labelled K-2, was prepared after Dr. Pfchov~ o f the Institute for Animal Production, Prague, and the tritium-labelled progesterone (Tracer: [1~, 2~ (n) -3 H]-progesterone, specific activity 1.59 TBq/mmol, cca 10 000 cpm/0.1 ml per test tube) was obtained from Great Britain (The Radiochemical Centre, Amersham). Unfixed (free) labelled progesterone was removed by adsorption with charcoal (norit A, produced by Serva, Heidelberg, FRG} and by centrifugation. The beta-radiation of tritium in the supernatant was measured by liquid scintillation in dioxan scintillation solution on the Packard beta-spectrometer. Three percent synthetic casein was used as equivalent medium for the formation of the calibration series after Pfchov~ et al. (1977). In variant A cows having ~< 1.4 ng progesterone per ml in their milk were regarded as non-pregnant and those with ~> 1.5 ng progesterone per ml were regarded as pregnant. In variants B and C the line dividing pregnancy from non-pregnancy was at a higher level, 2.4 ng progesterone per ml. In variant B cows with a milk progesterone level < 2.4 ng m1-1 in the first sampling and ~> 2.5 ng m1-1 in the second and third sampling were regarded as pregnant. Those which had a progesterone level > 2.4 ng m1-1 in the first sampling were regarded as inseminated outside oestrus. In variant C cows with a progesterone level ~> 2.5 ng ml -~ at all three samplings were considered as pregnant and those in which the progesterone level decreased at least once to < 2.4 ng m1-1 were considered as non-pregnant. The dividing lines of 1.4 ng mg -1 for milk samples obtained by foremilking and 2.4 ng ml -~ for milk samples obtained f r o m the whole a m o u n t o f milk from single milking had already been indicated as the best for the given m e t h o d o f sampling (Zaoral et al., 1982). In all cows pregnancy was diagnosed by rectal palpation between 60 and 90 days after insemination. The differences between the groups were subjected to the chi-square test after Myslivec (1957). RESULTS AND DISCUSSION No significant differences in the proportion of results in agreement in both tests (progesterone test and clinical examination) were f o u n d in the group o f non-pregnant cows between variants A and B (Tables I and II) and between the different combinations of milk sampling in variant B (Table II). It is interesting to note that in the separate evaluation of the third sampling
184 TABLE I Proportion of results in agreement in the PT and in clinical examination (milk samples on the 23rd day after insemination) -- variant A Group
Cows evaluated
Non-pregnant Pregnant Total
Results in agreement
504 1188 1691
Number
%
441 799 1240
87.7 67.3 73.3
TABLE II Proportion of results in agreement in the PT and in clinical determination (milk samples on day of insemination or on the 1st day, and on 19th and 23rd days after insemination) variant B
-
-
Sampling combination
Non-pregnant n
1, 2, 1, 1, 2 3
2, 3 3 2 3
60 50 63 48 40 37
Pregnant
Total
In agreement n
In agreement
No.
%
No.
%
51 43 51 42 33 34
85.0 86.0 81.0 87.5 82.5 91.9
74 80 80 81 96 102
78.7 70.8 69.0 73.6 58.2 67.1
94 113 116 110 165 152
n
154 163 179 158 205 189
In agreement No.
%
125 223 131 123 129 136
81.2 75.5 73.2 77.8 62.9 72.0
in variant B t h e highest p e r c e n t a g e o f results in a g r e e m e n t ( 9 1 . 9 % ) was obtained, even higher t h a n with t h e c o m b i n a t i o n o f t w o and t h r e e samplings (Table II). H o w e v e r , all t h e differences are statistically insignificant. In the g r o u p o f p r e g n a n t c o w s t h e p r o p o r t i o n o f the results in a g r e e m e n t in PT a n d in clinical e x a m i n a t i o n is a b o u t t h e same in variant A (Table I) a n d in the case o f single sampling in variant B (Table II). In t h e g r o u p o f p r e g n a n t c o w s t h e highest a g r e e m e n t b e t w e e n PT and clinical e x a m i n a t i o n was r e c o r d e d in t h e c o m b i n a t i o n o f t h r e e samplings ( 7 8 . 7 % ) w h i c h was significantly higher t h a n in t h e case o f separate s e c o n d sampling at P < 0.01 a n d t h a n in t h e t h i r d sampling at P < 0.05. T h e c o m b i n a t i o n o f t h e first a n d t h i r d sampling also h a d a significantly higher a g r e e m e n t o f results b e t w e e n PT and clinical e x a m i n a t i o n (73.6%) in c o m p a r i s o n w i t h t h e s e c o n d sampling at P < 0.01 a n d t h e c o m b i n a t i o n o f t h e s e c o n d and t h i r d sampling (70.8%) h a d a higher a g r e e m e n t in c o m p a r i s o n with t h e s e c o n d sampling at P < 0.05. T h e results in t h e g r o u p o f p r e g n a n t c o w s also i n f l u e n c e d the overall results (Table II). T h e highest a g r e e m e n t b e t w e e n t h e PT and clinical examina-
185 t i o n results was o b t a i n e d in t h e c o m b i n a t i o n o f s a m p l i n g s 1, 2 a n d 3 a n d this a g r e e m e n t was significantly d i f f e r e n t f r o m s e p a r a t e s a m p l i n g 2 at P 0.01, a n d f r o m s a m p l i n g 3 a t P < 0.05. In a d d i t i o n , t h e results o f s e p a r a t e s a m p l i n g 2 w e r e w o r s e t h a n in t h e c o m b i n a t i o n o f samplings 1 a n d 3 at P <= 0.01, a n d t h a n in t h e c o m b i n a t i o n s o f s a m p l i n g s 2 a n d 3 a n d o f 1 a n d a n d 2 at P ~ 0.05. Also in v a r i a n t C ( T a b l e I I I ) t h e d i f f e r e n c e s b e t w e e n all t h e c o m b i n a t i o n s in t h e g r o u p o f n o n - p r e g n a n t c o w s w e r e insignificant at P ~ 0.05. In t h e g r o u p o f p r e g n a n t c o w s t h e single s a m p l i n g s s h o w e d a significantly p o o r e r a g r e e m e n t o f results in c o m p a r i s o n w i t h all c o m b i n a t i o n s o f t w o samplings at P < 0 . 0 5 a n d in c o m p a r i s o n w i t h t h r e e - s a m p l i n g c o m b i n a t i o n s at P ~ 0.01. T h e significance o f t h e overall results was t h e same. A n increased n u m b e r o f s a m p l i n g s increases t h e p r o p o r t i o n o f c o r r e c t l y P T - d e t e r m i n e d n o n p r e g n a n t cows. H e n c e , t h e p r o p o r t i o n o f i n c o r r e c t l y P T - d e t e r m i n e d c o w s was r e d u c e d in t h e g r o u p o f p r e g n a n t cows. TABLE IH Proportion of results in agreement in the PT and in clinical examination (milk sampled three times after each insemination, sampling days only Monday and Thursday) -variant C Sampling combination
Non-pregnant n
1,2,3 1, 2 1,3 2,3 1 2
126 106 92 93 74 72
Pregnant
Total
In agreement n
In agreement
No.
%
No.
%
116 99 85 86 71 67
92.1 93.4 92.4 92.5 95.9 93.1
208 221 217 217 226 223
83.9 78.6 78.9 78.3 70.6 71.2
248 281 275 277 320 313
n
374 387 367 370 394 385
In agreement No.
%
324 320 302 303 297 290
86.6 82.7 82.3 81.9 75.4 75.3
O u r results c o n f o r m t o t h e c l a i m b y Giinzler et al. ( 1 9 7 5 ) t h a t 90% agreem e n t b e t w e e n t h e results in t h e p r o g e s t e r o n e t e s t a n d in clinical e x a m i n a t i o n can b e r e g a r d e d as high a n d c a n n o t b e f u r t h e r i m p r o v e d . C o n t r a r y t o t h e d a t a o f H e a p et al. ( 1 9 7 6 ) , o u r tests s h o w e d t h a t t h e c o m b i n a t i o n o f t w o s a m p l e s was m o r e a d v a n t a g e o u s a n d t h a t in v a r i a n t C this c o m b i n a t i o n gave a statistically significantly higher a g r e e m e n t o f results t h a n w i t h single m i l k sampling. V a r i a n t C was d e m o n s t r a t e d t o give results c o m p a r a b l e t o t h o s e in v a r i a n t B; t h e l a b o r i o u s d a i l y m i l k s a m p l i n g t h e r e f o r e a p p e a r s u n n e c e s s a r y . R e m a r k a b l e results w e r e o b t a i n e d in e x a m i n i n g t h e p r o p o r t i o n o f t h e PTd e t e r m i n e d n o n - p r e g n a n t c o w s ( p r o g e s t e r o n e <~ 2.4 ng m1-1) o f t h e t o t a l n u m b e r o f clinically d e t e r m i n e d n o n - p r e g n a n t c o w s ( T a b l e IV). T h e c o m p a r a t i v e l y l o w p e r c e n t a g e o f t h e n o n - p r e g n a n t c o w s f o u n d in t h e first sampl-
186 TABLE IV Proportion of congruent results in non-pregnant cows -- variant C Sampling or sampling cornbination
1 2 1 and 2 1 and 3 2 and3 1, 2 and 3
Non-pregnant total
165 157 159 143 146 156
Of this, in group Up to 2.4 ng m1-1
Above 2.4 ng m1-1
No.
%
No.
%
71 67 99 85 86 116
43.0 42.7 62.3 59.4 58.9 74.4
94 90 60 58 60 40
57.0 57.3 37.7 40.6 41.1 25.6
ing (43.0% or 4 2 . 7 % ) was i m p r o v e d b y t h e c o m b i n a t i o n o f t w o samplings b y 1 5 - - 2 0 % , a n d b y t h e c o m b i n a t i o n o f t h r e e samplings b y a n o t h e r 15%. T h e influence o f an increased n u m b e r o f samplings is statistically h i g h l y significant (Table V). T h a t is t o say, an increased n u m b e r o f samplings reduces the n u m b e r o f n o n - p r e g n a n t c o w s i n c o r r e c t l y d e t e r m i n e d b y t h e p r o g e s t e r o n e test. The e v a l u a t i o n o f t h e results in variant B revealed t h a t 14.7% o f t h e inseminations were p e r f o r m e d o u t s i d e oestrus. This c o r r e s p o n d s t o t h e earlier finding described b y Elsaesser ( 1 9 7 9 ) . TABLE V Proportion of congruent results in non-pregnant cows, x 2 test, variant C
1 2 1 and 2 1 and 3 2 and 3 1,2 and 3
1
2
1 and 2
1 and 3
2 and 3
1, 2 and 3
--
0.041 --
12.0118"* 12.1556"* --
8.2536** 8.4170"* 0.2519 --
7.8069** 7.9727** 0.3598 0.0086 --
32.3667** 32.3507** 5.3150" 7.5349** 8.1341"*
*P < 0.05, **P < 0.01. ACKNOWLEDGEMENTS T h e a u t h o r s are obliged t o Dr. D. Pichovfi and t o Dr. J. P / c h a f o r advice in familiarizing w i t h t h e r a d i o i m m u n o l o g i c a l m e t h o d , f o r special consultat i o n s and f o r p r o v i d i n g t h e specific a n t i b o d y . T h a n k s also go t o Mrs. M. N~mcovfi and Mr. P. B a n ~ f o r t e c h n i c a l assistance.
187 REFERENCES Booth, J.M. and Holdsworth, R.J., 1976. The establishment and operation of a central laboratory for pregnancy testing in cows. Br. Vet. J., 132: 518--524. Elsaesser, F., 1979. In welcher Weise kann die Milch-Progesteron-Bestimmung zur Verbesserung der Fortpflanzungsteuerung beim Rind beitragen? Tierziiehter, 31: 94--95. Gfinzler, O., KorndSrfer, L., Lohoff, H., Hamburger, R. and Hoffmann, B., 1975. Praktische Erfahrungen mit der Progesteron-Bestimmung in der Milch zur Erfassung des Fertilit~/tszustandes bei der Kuh. Tier'drztl. Umsch., 30 : 111--118. Heap, R.B., Holdsworth, R.J., Gadsby, J.E., Laing, J.A. and Walters, D.E., 1976. Pregnancy diagnosis in the cow from milk progesterone concentration. Br. Vet. J., 132: 445--446. Hoffmann, B., Hamburger, R., Giinzler, O. KorndSrfer, L. and Lohoff, H., 1974. Determination of progesterone in milk applied for pregnancy diagnosis in the cow. Theriogenology, 2: 21--28. Myslivec, V., 1957. Statistick~ Metody Zem~d~lsk~ho a Lesnick~ho V~zkumnictvf. (Statistical Methods o f Agricultural and Sylvicultural Research). CSAZV, Prague, 555 pp. Pfchov~, D., Pfcha, J. Stupnicki, R., Krupkov~, B. and Polz, Z., 1977. Direct radioimmunological determination of progesterone in milk. Sci. Agric. Bohemoslov., 9: 243--251. Shemesh, M., Ayalon, N., Shalev, E., Nerya, A., Schindler, H. and Milguir, F., 1978. Milk progesterone measurement in dairy cows: correlation with oestrus and pregnancy determination. Theriogenology, 9: 343--352. Wishart, L.F., Head, V.A. and Hort, C.E., 1975. Early pregnancy diagnosis in cattle. Vet. Rec., 96: 34--38. Zaoral, J., PSschl, M. and Mi~'~nek, H., 1982. Progesteronov~ test zab~ez~v~nf. (Progesterone test in conception diagnosis). Zivocisn~ V~roba, 27 (1): 7--13.
181
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREGNANCY TESTING OF COWS BY DETERMINING LEVELS IN MILK
PROGESTERONE
J. Z A O R A L , M. P()SCHL* and J. KVAPILIK
Research Institute for Cattle Breeding, Rapotih 788 13 Vik~ovice (Czechoslovakia) *State Breeding Enterprises, General Directorate 252 O0 Hradi~tko pod Medn[kem (Czechoslovakia) (Accepted 30 June 1981)
ABSTRACT Zaoral, J., PSschl, M. and Kvapilfk, J., 1982. Pregnancy testing of cows by determining progesterone levels in milk. Anita. Reprod. Sci., 4: 181--187. In six herds, each comprising 400 to 600 cows, the progesterone test (PT) was compared at different numbers of milk samplings from one cow and at a reduced number of days of milk sampling per week (from seven to two per week). There were three variants. In four herds the foremilk was sampled on the 23rd day after insemination in the period from October 1978 to September 1979 (variant A). In the remaining two herds the samples were taken from the whole amount of milk from single milking in May to July 1979 (variant B) and in August to November 1979 (variant C). In variant B the samples were taken on the day of insemination or the first day after insemination, and on the 19th and 23rd days after insemination. In variant C also three samples were taken after each insemination, but only on two days in the week, Monday and Thursday ( l s t sampling 19--22, 2nd sampling 22--25 and 3rd sampling 29--32 days after insemination). A radioimmunological method was used to determine progesterone (tritium-labelled progesterone). Pregnancy was diagnosed by rectal palpation between the 60th and 90th days after insemination. In comparison with a single sampling, the use of two or three samples after insemination in variants B and C did not improve the proportion of correct results in the group of non-pregnant cows (progesterone level ~< 2.4 ng m1-1) nor in the overall results. Of the clinically identified non-pregnant cows, single milk sampling correctly determined 42.7 and 43.0%, the combination of two samplings 58.9 to 62.3%, and the combination o f three samplings 74.7% of the cows. The differences were significant (P < 0.01). The reduction of sampling days from seven to two in a week did not influence adversely the proportion of the results which were in agreement in the PT and clinical examination.
INTRODUCTION The direct radioimmunological determination of milk progesterone makes it possible to use this m e t h o d for d e t e r m i n i n g the success o f i n s e m i n a t i o n . T h i s is o f p a r t i c u l a r i m p o r t a n c e f o r l a r g e - c a p a c i t y c o w - h o u s e s in w h i c h t h e p r o c e s s o f r e p r o d u c t i o n is a r a t h e r d i f f i c u l t t a s k . I f t h i s m e t h o d is u s e d , t h e
0378-4320/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
182 service period is not unnecessarily prolonged and hence economic losses are prevented. The m e t h o d is particularly suitable for herds included in a controlled breeding programme (Wishart et al., 1975). An important prerequisite for a practical use of the progesterone test is the proportion of the results of this test which agree with the results of clinical examination. The majority of authors believe that non-pregnancy can be predicted with an accuracy of 95--100% and pregnancy with an accuracy of 75--85% (Hoffmann et al., 1974; Heap et al., 1976; Shemesh et al., 1978). Hence generally correct results can be expected in about 80% of the animals, and with two measurements (on the day of insemination and the 20th day after insemination) in 9 0 % of the animals. These results should be regarded as highly accurate and practically cannot be further improved (Gilnzler et al., 1975). Sampling on the day of insemination can eliminate inseminations performed at the wrong time. The proportion of such inseminations is 10--20% (Elsaesser, 1979). Paired sampling did n o t show a statistically significant improvement of the results in comparison with single samples (Heap et al., 1976). The combination of sampling on the 21st and 24th days after insemination is regarded as the best (the following sampling terms were used by Heap et al.: the 21st, 24th, 28th and 42nd days). In commercial service,rendered in Great Britain by the Milk Marketing Board, milk sampling is repeated on the 42r/~1 day if the results of sampling on the 24th day are dubious (Booth and Holdsworth, 1976). In this paper, single milk sampling is compared with double and/or triple samplings under farm conditions. The facilitation of samplings by reducing the number of sampling days per week is also discussed. MATERIAL AND METHODS A pilot experiment was conducted in six herds of 400 to 600 cows in each (mostly crossbreds between the Bohemian Red-Spotted and Holstein breeds). The experiment had three variants. Variant A -- in four herds foremilk was sampled on the 23rd day after insemination in the period from October 1978 to September 1979. In the remaining two herds milk was sampled from the whole a m o u n t of milk obtained in single milking. These two herds comprised variant B -- from May to July 1979 -- and variant C -- from August to November 1979. Variant B -- three samplings performed after each insemination. The first sample was taken on the day of insemination or one day later. The second sample was taken 19 days after insemination and the third sampling followed on the 23rd day after insemination. Variant C -- three samplings were performed after each insemination. The sampling days were two in a week (Monday and Thursday). The first sample was taken 19--22 days after insemination and the second was taken on Thursday if the first sampling had been performed on Monday and vice versa. The second sampling took place 22--25 days after insemination and the third
183 followed after 7 days, i.e. 29--32 days after insemination. A b o u t 2 ml milk was preserved with potassium dichromate and was kept in a refrigerator until dispatch or transport to the laboratory. The samples were sent by mail or transported by car to the laboratory once or twice a week. The radioimmUnological m e t h o d (RIA) with direct progesterone determination from whole milk after Pfchov~ et al. (1977) was used for the analysis of the milk samples. Milk (20 pl) was diluted with buffer (1 : 4) and then assayed. The specific a n t i b o d y used, labelled K-2, was prepared after Dr. Pfchov~ o f the Institute for Animal Production, Prague, and the tritium-labelled progesterone (Tracer: [1~, 2~ (n) -3 H]-progesterone, specific activity 1.59 TBq/mmol, cca 10 000 cpm/0.1 ml per test tube) was obtained from Great Britain (The Radiochemical Centre, Amersham). Unfixed (free) labelled progesterone was removed by adsorption with charcoal (norit A, produced by Serva, Heidelberg, FRG} and by centrifugation. The beta-radiation of tritium in the supernatant was measured by liquid scintillation in dioxan scintillation solution on the Packard beta-spectrometer. Three percent synthetic casein was used as equivalent medium for the formation of the calibration series after Pfchov~ et al. (1977). In variant A cows having ~< 1.4 ng progesterone per ml in their milk were regarded as non-pregnant and those with ~> 1.5 ng progesterone per ml were regarded as pregnant. In variants B and C the line dividing pregnancy from non-pregnancy was at a higher level, 2.4 ng progesterone per ml. In variant B cows with a milk progesterone level < 2.4 ng m1-1 in the first sampling and ~> 2.5 ng m1-1 in the second and third sampling were regarded as pregnant. Those which had a progesterone level > 2.4 ng m1-1 in the first sampling were regarded as inseminated outside oestrus. In variant C cows with a progesterone level ~> 2.5 ng ml -~ at all three samplings were considered as pregnant and those in which the progesterone level decreased at least once to < 2.4 ng m1-1 were considered as non-pregnant. The dividing lines of 1.4 ng mg -1 for milk samples obtained by foremilking and 2.4 ng ml -~ for milk samples obtained f r o m the whole a m o u n t o f milk from single milking had already been indicated as the best for the given m e t h o d o f sampling (Zaoral et al., 1982). In all cows pregnancy was diagnosed by rectal palpation between 60 and 90 days after insemination. The differences between the groups were subjected to the chi-square test after Myslivec (1957). RESULTS AND DISCUSSION No significant differences in the proportion of results in agreement in both tests (progesterone test and clinical examination) were f o u n d in the group o f non-pregnant cows between variants A and B (Tables I and II) and between the different combinations of milk sampling in variant B (Table II). It is interesting to note that in the separate evaluation of the third sampling
184 TABLE I Proportion of results in agreement in the PT and in clinical examination (milk samples on the 23rd day after insemination) -- variant A Group
Cows evaluated
Non-pregnant Pregnant Total
Results in agreement
504 1188 1691
Number
%
441 799 1240
87.7 67.3 73.3
TABLE II Proportion of results in agreement in the PT and in clinical determination (milk samples on day of insemination or on the 1st day, and on 19th and 23rd days after insemination) variant B
-
-
Sampling combination
Non-pregnant n
1, 2, 1, 1, 2 3
2, 3 3 2 3
60 50 63 48 40 37
Pregnant
Total
In agreement n
In agreement
No.
%
No.
%
51 43 51 42 33 34
85.0 86.0 81.0 87.5 82.5 91.9
74 80 80 81 96 102
78.7 70.8 69.0 73.6 58.2 67.1
94 113 116 110 165 152
n
154 163 179 158 205 189
In agreement No.
%
125 223 131 123 129 136
81.2 75.5 73.2 77.8 62.9 72.0
in variant B t h e highest p e r c e n t a g e o f results in a g r e e m e n t ( 9 1 . 9 % ) was obtained, even higher t h a n with t h e c o m b i n a t i o n o f t w o and t h r e e samplings (Table II). H o w e v e r , all t h e differences are statistically insignificant. In the g r o u p o f p r e g n a n t c o w s t h e p r o p o r t i o n o f the results in a g r e e m e n t in PT a n d in clinical e x a m i n a t i o n is a b o u t t h e same in variant A (Table I) a n d in the case o f single sampling in variant B (Table II). In t h e g r o u p o f p r e g n a n t c o w s t h e highest a g r e e m e n t b e t w e e n PT and clinical e x a m i n a t i o n was r e c o r d e d in t h e c o m b i n a t i o n o f t h r e e samplings ( 7 8 . 7 % ) w h i c h was significantly higher t h a n in t h e case o f separate s e c o n d sampling at P < 0.01 a n d t h a n in t h e t h i r d sampling at P < 0.05. T h e c o m b i n a t i o n o f t h e first a n d t h i r d sampling also h a d a significantly higher a g r e e m e n t o f results b e t w e e n PT and clinical e x a m i n a t i o n (73.6%) in c o m p a r i s o n w i t h t h e s e c o n d sampling at P < 0.01 a n d t h e c o m b i n a t i o n o f t h e s e c o n d and t h i r d sampling (70.8%) h a d a higher a g r e e m e n t in c o m p a r i s o n with t h e s e c o n d sampling at P < 0.05. T h e results in t h e g r o u p o f p r e g n a n t c o w s also i n f l u e n c e d the overall results (Table II). T h e highest a g r e e m e n t b e t w e e n t h e PT and clinical examina-
185 t i o n results was o b t a i n e d in t h e c o m b i n a t i o n o f s a m p l i n g s 1, 2 a n d 3 a n d this a g r e e m e n t was significantly d i f f e r e n t f r o m s e p a r a t e s a m p l i n g 2 at P 0.01, a n d f r o m s a m p l i n g 3 a t P < 0.05. In a d d i t i o n , t h e results o f s e p a r a t e s a m p l i n g 2 w e r e w o r s e t h a n in t h e c o m b i n a t i o n o f samplings 1 a n d 3 at P <= 0.01, a n d t h a n in t h e c o m b i n a t i o n s o f s a m p l i n g s 2 a n d 3 a n d o f 1 a n d a n d 2 at P ~ 0.05. Also in v a r i a n t C ( T a b l e I I I ) t h e d i f f e r e n c e s b e t w e e n all t h e c o m b i n a t i o n s in t h e g r o u p o f n o n - p r e g n a n t c o w s w e r e insignificant at P ~ 0.05. In t h e g r o u p o f p r e g n a n t c o w s t h e single s a m p l i n g s s h o w e d a significantly p o o r e r a g r e e m e n t o f results in c o m p a r i s o n w i t h all c o m b i n a t i o n s o f t w o samplings at P < 0 . 0 5 a n d in c o m p a r i s o n w i t h t h r e e - s a m p l i n g c o m b i n a t i o n s at P ~ 0.01. T h e significance o f t h e overall results was t h e same. A n increased n u m b e r o f s a m p l i n g s increases t h e p r o p o r t i o n o f c o r r e c t l y P T - d e t e r m i n e d n o n p r e g n a n t cows. H e n c e , t h e p r o p o r t i o n o f i n c o r r e c t l y P T - d e t e r m i n e d c o w s was r e d u c e d in t h e g r o u p o f p r e g n a n t cows. TABLE IH Proportion of results in agreement in the PT and in clinical examination (milk sampled three times after each insemination, sampling days only Monday and Thursday) -variant C Sampling combination
Non-pregnant n
1,2,3 1, 2 1,3 2,3 1 2
126 106 92 93 74 72
Pregnant
Total
In agreement n
In agreement
No.
%
No.
%
116 99 85 86 71 67
92.1 93.4 92.4 92.5 95.9 93.1
208 221 217 217 226 223
83.9 78.6 78.9 78.3 70.6 71.2
248 281 275 277 320 313
n
374 387 367 370 394 385
In agreement No.
%
324 320 302 303 297 290
86.6 82.7 82.3 81.9 75.4 75.3
O u r results c o n f o r m t o t h e c l a i m b y Giinzler et al. ( 1 9 7 5 ) t h a t 90% agreem e n t b e t w e e n t h e results in t h e p r o g e s t e r o n e t e s t a n d in clinical e x a m i n a t i o n can b e r e g a r d e d as high a n d c a n n o t b e f u r t h e r i m p r o v e d . C o n t r a r y t o t h e d a t a o f H e a p et al. ( 1 9 7 6 ) , o u r tests s h o w e d t h a t t h e c o m b i n a t i o n o f t w o s a m p l e s was m o r e a d v a n t a g e o u s a n d t h a t in v a r i a n t C this c o m b i n a t i o n gave a statistically significantly higher a g r e e m e n t o f results t h a n w i t h single m i l k sampling. V a r i a n t C was d e m o n s t r a t e d t o give results c o m p a r a b l e t o t h o s e in v a r i a n t B; t h e l a b o r i o u s d a i l y m i l k s a m p l i n g t h e r e f o r e a p p e a r s u n n e c e s s a r y . R e m a r k a b l e results w e r e o b t a i n e d in e x a m i n i n g t h e p r o p o r t i o n o f t h e PTd e t e r m i n e d n o n - p r e g n a n t c o w s ( p r o g e s t e r o n e <~ 2.4 ng m1-1) o f t h e t o t a l n u m b e r o f clinically d e t e r m i n e d n o n - p r e g n a n t c o w s ( T a b l e IV). T h e c o m p a r a t i v e l y l o w p e r c e n t a g e o f t h e n o n - p r e g n a n t c o w s f o u n d in t h e first sampl-
186 TABLE IV Proportion of congruent results in non-pregnant cows -- variant C Sampling or sampling cornbination
1 2 1 and 2 1 and 3 2 and3 1, 2 and 3
Non-pregnant total
165 157 159 143 146 156
Of this, in group Up to 2.4 ng m1-1
Above 2.4 ng m1-1
No.
%
No.
%
71 67 99 85 86 116
43.0 42.7 62.3 59.4 58.9 74.4
94 90 60 58 60 40
57.0 57.3 37.7 40.6 41.1 25.6
ing (43.0% or 4 2 . 7 % ) was i m p r o v e d b y t h e c o m b i n a t i o n o f t w o samplings b y 1 5 - - 2 0 % , a n d b y t h e c o m b i n a t i o n o f t h r e e samplings b y a n o t h e r 15%. T h e influence o f an increased n u m b e r o f samplings is statistically h i g h l y significant (Table V). T h a t is t o say, an increased n u m b e r o f samplings reduces the n u m b e r o f n o n - p r e g n a n t c o w s i n c o r r e c t l y d e t e r m i n e d b y t h e p r o g e s t e r o n e test. The e v a l u a t i o n o f t h e results in variant B revealed t h a t 14.7% o f t h e inseminations were p e r f o r m e d o u t s i d e oestrus. This c o r r e s p o n d s t o t h e earlier finding described b y Elsaesser ( 1 9 7 9 ) . TABLE V Proportion of congruent results in non-pregnant cows, x 2 test, variant C
1 2 1 and 2 1 and 3 2 and 3 1,2 and 3
1
2
1 and 2
1 and 3
2 and 3
1, 2 and 3
--
0.041 --
12.0118"* 12.1556"* --
8.2536** 8.4170"* 0.2519 --
7.8069** 7.9727** 0.3598 0.0086 --
32.3667** 32.3507** 5.3150" 7.5349** 8.1341"*
*P < 0.05, **P < 0.01. ACKNOWLEDGEMENTS T h e a u t h o r s are obliged t o Dr. D. Pichovfi and t o Dr. J. P / c h a f o r advice in familiarizing w i t h t h e r a d i o i m m u n o l o g i c a l m e t h o d , f o r special consultat i o n s and f o r p r o v i d i n g t h e specific a n t i b o d y . T h a n k s also go t o Mrs. M. N~mcovfi and Mr. P. B a n ~ f o r t e c h n i c a l assistance.
187 REFERENCES Booth, J.M. and Holdsworth, R.J., 1976. The establishment and operation of a central laboratory for pregnancy testing in cows. Br. Vet. J., 132: 518--524. Elsaesser, F., 1979. In welcher Weise kann die Milch-Progesteron-Bestimmung zur Verbesserung der Fortpflanzungsteuerung beim Rind beitragen? Tierziiehter, 31: 94--95. Gfinzler, O., KorndSrfer, L., Lohoff, H., Hamburger, R. and Hoffmann, B., 1975. Praktische Erfahrungen mit der Progesteron-Bestimmung in der Milch zur Erfassung des Fertilit~/tszustandes bei der Kuh. Tier'drztl. Umsch., 30 : 111--118. Heap, R.B., Holdsworth, R.J., Gadsby, J.E., Laing, J.A. and Walters, D.E., 1976. Pregnancy diagnosis in the cow from milk progesterone concentration. Br. Vet. J., 132: 445--446. Hoffmann, B., Hamburger, R., Giinzler, O. KorndSrfer, L. and Lohoff, H., 1974. Determination of progesterone in milk applied for pregnancy diagnosis in the cow. Theriogenology, 2: 21--28. Myslivec, V., 1957. Statistick~ Metody Zem~d~lsk~ho a Lesnick~ho V~zkumnictvf. (Statistical Methods o f Agricultural and Sylvicultural Research). CSAZV, Prague, 555 pp. Pfchov~, D., Pfcha, J. Stupnicki, R., Krupkov~, B. and Polz, Z., 1977. Direct radioimmunological determination of progesterone in milk. Sci. Agric. Bohemoslov., 9: 243--251. Shemesh, M., Ayalon, N., Shalev, E., Nerya, A., Schindler, H. and Milguir, F., 1978. Milk progesterone measurement in dairy cows: correlation with oestrus and pregnancy determination. Theriogenology, 9: 343--352. Wishart, L.F., Head, V.A. and Hort, C.E., 1975. Early pregnancy diagnosis in cattle. Vet. Rec., 96: 34--38. Zaoral, J., PSschl, M. and Mi~'~nek, H., 1982. Progesteronov~ test zab~ez~v~nf. (Progesterone test in conception diagnosis). Zivocisn~ V~roba, 27 (1): 7--13.