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A New Approach to the Use of Progesterone Levels for Pregnancy Determination
Br. uel. J. ( 1983). 139, 41
A NE\,Y APPROACH TO THE USE OF PROGESTERONE LEVELS FOR PREGNANCY DETERMINATION
BY M . SHEMESH, N. AVALON, S . LA VI, F. Mll.ECU IR , L. S. SHORE AND D . T OBY
Departments of Hormone Research and Reproduction, Ki1nron Veterinary Institute, Beil Dagan, P.O.B. 12, Israel
SUMMARY Intravaginal sponges impregna ted with 250 m g m ed roxyprogeste ro ne (MA P ) were in serted in Israe li Friesian cows a nd th e effect o n pl as m a progesterone a nd ea rly pregnancy di ag nosis based on milk progeste ron e o bse rved . It was found that:( I ) exogeno us MAP prevented the formati o n of a norm a l corpu s luteum if it was a dmini s tered for a number of d ays starting o n the d ay of oestrus. (2) In contrast, exoge no us MAP did not interfere with the d eve lopme nt of a normal corpus lute um wh en it was applied six or more d ays after oestrus. (3) R e m ova l of the MAP a fter the 17th d ay following oestrus resulted in a rapid fa ll in pl as m a a nd milk progesterone in non- pregna nt cows which (4) a ll owed a compl ete ly accurate determination o f pregna ncy to be m ade, e limin ati ng the 20% fa lse posi tives which us ua lly occur. (5) Fertility was not a dverse ly a ffected and (6) m ay act ua lly have been improved, es pecially in herd s with low fertility due to hig h con centra tion s of phyto-oest rogens in the feed .
INTRODUCT ION The d ay-to-d a y vanat1ons in pl as m a progesteron e in non-pregna nt cows display a cyclic p a ttern (Fig. I) ch aracterized by (a) minimal plasm a progeste ro n e levels, <0·5 ng/ml , fro m two d ays before to three days after ovulation, ( b) a sh a rp rise fro m day 5 to day 7 of th e cycl e, and (c) a very slow rise or platea u o n the s ubsequ ent eight to I 0 days, Ou c tu a ting about a m ean of 4 to 5 ng/ml. An importa nt fea ture of the bovine cycle is the abrupt fa ll in periphera l plasma progestero ne levels which occurs be tween th e third and second day before oes trus. Since thi s fall is not seen in pregna nt a nimals , an ea rl y pregn ancy diagnosis can be made at this time by progesterone determinations in plasm a (S hemesh, Ayalon & Lindn er, 1968; Pope et al., 1969; Sta benfeldt, Ewing & McDo na ld , 1969; Donaldson, Basse tt & Thorburn , 1970; Robertson, 1972) as well as in milk (Laing & H ea p, 1971; H ea p et al., 1973 ; Hoffm a n & Hamburger, 1973; Darling, Laing & Harkn ess, 1974;
BRITISH VETERINARY JO U RNA L, 139, I
42
Hoffm an et al., 1974; Thibier, 1974; Dobson, Midmer & Fitzpatrick, 1975; Ball & Pope, 1976; Shemesh et al., 1978). It is possible to use milk progesterone determin a tions since milk progesteron e accurately refle cts the pl as ma co ncentration of progeste rone (T ab le I).
7
6-
3-
{Oest r us)
Days after oestrus
Fig. I. Periph era l plasma progesterone levels in cows during the oes trous cycle (TLC/C PB meth od) . Cycle length 20 to 22 days, vert ica l bars indicate ± S£ (from Shemesh, Lindn er & A)•a lon, 197 1).
TABLE !
PROGEST ERON E CONCENTRAT IO N I N FORE M ILK AND JUGULAR PLASMA IN PREGNANT AND NON-PREGNANT CO WS AT 24 DAYS AFTER I NSEMINAT ION ( FROM SHEMESH ETAL., 1978)
Foremilk unextracted
Proges terone ng/ ml ± SEM n
Foremilk fat free
jugular plasma
Pregnant
Not pregnant
Pregnant
Not pregnant
Pregnant
Not pregnant
4· 73 ± 2·2 44
0·1-0·6 41
2·68 ± 1·8 21
0·1 29
2·9 ± 0·9 21
0·1-0·3 12
43
PROGESTERONE AND PREG NANCY DETERMINATION TABLE II
ACCURACY OF PREGNANCY DIAGNOSIS BY MILK PROGESTERONE AT 24 DAYS AFTER INSEMINATION (F ROM SHEMESH ET AL., 1978)
II
85
Diagnosed correctly pregnant
Diagnosed correctly not pregnant
No .
%
No.
%
34/44
78
41/41
100
Diagnosis of non-pregnancy can be routinely performed with 95 to 100% accuracy. However, when making a positive diagnosis only 70 to 80% accuracy has be en achieved (Table II); this error may be due to either lutea l phase progesteron e or early embryonic mortality. In an attempt to improve the accuracy of the test, progestinimpregnated vaginal sponges (PIVS) were applied to reduce the widespread range in the time of return to oestrus seen in insemin a ted cows which are not pregnant (Shemesh et. al. , 1981). The efficacy of the PIVS in inducing oestrus has been previously described (Ayalon & Marcus, 1975). Furthermore, in order to elucidate the mode of act ion of this exogenous progesterone, the early effect of PIVS on periph era l plasma progesterone at different periods of the oestrous cycle and following insemination was also st udi ed .
MATER IALS AND METHODS
Animals Experiment 1. Israeli Friesian heifers from a well-managed commercial herd were
housed in our Institute for three months. Animals were synchronized with Estrumate a nd on the day of oestrus, PIVS impregnated with 250 mg medroxyprogesterone (MAP) were inserted in five cows and withdrawn after 10 days. Jugular venous samples were taken a day before the ex pected oestrus (36 h after Estrumate) and on each of the following 12 days. Experiment 2. Nine heifers were inseminated and PIVS impregnated with MAP were inserted on day 6 after insemination (p.i.) and withdrawn on day 17.Jug ul ar venous blood samples were taken on day 6 p.i . and on each of the subseq uent 17 days. Experiment 3. The cows were Israeli Friesian cattle in well-managed commercial herds from the centra l plains area. Oestrus was detected by twice-daily observation. Six to seven days following routine artificial insemination animals either had PIVS inserted or were left untreated. The sponges were removed on day 17 p.i . Milk sa mples were taken 21 to 24 days after oestrus for pregnancy determinations. Experiment 1. Twenty-four dairy cows were used from a herd with poor fertility associated with high levels ofphyto-oestrogen. Thirteen cows had PIVS impregnated with MAP inserted and eleven served as controls . Animals which did not become pregnant following the first insemination were inseminated a second time.
44
BRI T I SH V ETERIN A RY J OU RNA L , 139, I
Measurement of plasma and milk progesterone concentrations T he m easu re ment of pl as ma a nd milk proges te rone conce ntrations was perform ed as previo us ly d escribed (Sh em es h , Aya lon & M azo r, 1979). C lini ca l co nfirm at ion of pregna ncy was based on recta l pa lpa ti on six to seven weeks post in se min a ti on .
RESU LTS
Experiment I. Exoge no us M A P in se rted imra vagin a ll y on th e d a y of oes tru s res ulted in non-d e tec table va lu es of proges tero ne throug hou t th e period studi ed ( <0· 1 ng/ml ). Th e M AP a ppa rentl y d id not cross- react with proges terone wh en th e specifi c a nti bod y aga in st progesterone- I Ia hemi succin a te in the presen t stud y was used . Exj;eriment 2. In se rtio n of M A P on d ay 6 aft er oes tru s, when th e co rpu s lute u m was a lread y es ta blished, did not a ffec t th e lutea l ph ase progeste rone in eith er p reg na nt (n =6) or non- pregna nt (n =3) heifers (Fi g. 2). Th e level of pl as ma proges terone in th e pregna n t heife rs was slightl y hi gh er th a n th a t of th e non-preg na nt heifers. H owever, within 24 h a fte r MAP withdra wa l, a ll three non-preg na nt heifers displayed a
.....__.... Pregnant
0 --{)
Non- pre gnant
Intra -vag inal
MAP
E
.....
(n =6l
"'c: c: e"'
~ V>
"'0 "'
a.
0
E
V>
0
a:
Days
fi g . 2. Th e effect o f intravagin a l s po nges impregna ted \vith M A P o n periph e ra l pl as ma proges tero ne level s wh en in se rted on d ay six a ft e r in semin a ti o n.
PROGESTERONE AND PREGNANCY DETERMINATION
45
dramatic decline in their plasma progesterone conce ntrations (from 4 to~ · 5 ng/ml) . Furthermore, within 62 h the plasma progesterone was less than 0·5 ng/ml and all of the heifers returned to oestrus . Experiment 3. The results of the laboratory diagnosis, co nfirmed by rectal palpation, are presented in Table II I. The accuracy of the test for the absence of pregnancy in cows 21 to 24 days after oestrus was I 00%, regardless of wheth er PIVS were used or not. However, markedly different results were found in positive pregnancy diagnosis. In co ntras t to the control group, in which 11 of the 52 {21%) cows were incorrec tly diagnosed as pregnant, all 30 cows with PIVS were correctly diagnosed as pregnan t. The fertility rate in the control group was 43% a nd in the PIVS group it was 60%. This indicates a possible beneficial effect on fertility .
TAI3LEIIJ ACCURACY Or PREGNAN CY DIAGNOSIS BY MILK PROGESTERONE AT 21 TO 24 DAYS AFTER OESTRUS WITH AND WITHOUT THE USE Or PIVS. PREGNANCY WAS CONFIRMED AT SIX TO SEVEN \'\'EE KS BY RECTAL PALPATION (FROM SHEMESH ETA!.. , 1981)
Treatment
No. of cows
Diagnosed correctly fmgnant
Diagnosed correctly not pregnant
No.
%
No.
%
79 100
44/44 20/20
100 100
None
96
41/52
PIVS
50
30/30
TABLE IV INFLUENCE OF MAP ON CONCEPTION RATE IN A DAIRY HERD \-\1 1TH POOR FERTILITY ASSOCIATED WITH HIGH LEVELS OF PHYTO-OESTROGENS. PREGNANCY WAS CONFIRMED AT SIX TO SEVEN WEEKS BY RECTA L PALPATIO
Treatment
Conception rate
No. of cows 1st insemination
None
11
PIVS
13
1
2nd insemination
Total
No .
%
No .
%
No.
2/11 4/13
18
31
0/9 3/9
33
2/11 7/13
18 54
Experiment 4. The rate of conception in these sub-fertile cows with or without PIVS is shown in Table IV . The cows with PIVS had a 54% conception rate compared with 18% in the controls. This herd had clinical indications- high incidence of follicular cysts, nymphomania and short intervals between oestrous periods - that the infertility was due to high leve ls ofphyto-oestrogens in the feed . In the laboratory, it
46
BRITISH VETERINARYJOURNAL, 139, I
was found that the level of phyto-oestrogens in the various feed constituents ranged from the equivalent of 61 to 448 ng oestradiol/g dry matter as determined by radioreceptor assay (Shemesh, Lindner & Ayalon, 1973) and in-vitro bioassay (Kaplanski, Shemesh & Berman, 1981). DISCUSSION
Value of progesterone determinations in milk and plasmafor pregnancy detennination The determination of plasma progesterone gives an accurate picture of the status of the corpus luteum in the cow, i.e., following luteolysis, plasma progesterone falls rapidly to non-detectable levels. Defatted milk also accurately reflects the plasma concentration of progesterone. This study shows that l 00% correct pregn a ncy diagnosi s can be obtained if exogenous progesterone (MAP) is administered during the post-insemination period. Effect of exogenous MAP on plasma progesterone concentrations Exogenous MAP given on the day prior to expected oestrus prevented the form at ion of the corpus luteum as reflected by the absence oflutea l progesterone. The underlying mechanism involved is most probably connected with the fact that progesterone acts to suppress pituitary LH release in th e non-pregnant cow (Hansel et al., 1975 ). However, once the corpus luteum was established, MAP did not affect the plasm a progesterone concentration. The MAP did not therefore interfere with the functional corpus luteum. Indeed , the slightly higher proges terone concentrations associated with corpora lutea in the presence of a conceptus which have been reported by others (Hendricks et al., 1971; Erb et al., 1976; H anse l & Lukaszewska, personal communication) were seen in this study even when MAP was present. Effect of PI VS on improving the accuracy of milk progesterone for early pregnancy diagnosis In 1965, Robinson reported a new approach to oestrus synchronization in sheep using vaginal sponges to which a progestin compound was adsorbed. The method is widely used for sheep but in cattle it has not been commercially successful since experiments have not given uniform resu lts and many catt le failed to retain the sponge. However, Ayalon & Marcus ( 1975) have obtained l 00% retention rates using a locally-produced intravaginal sponge. The use of exogenous progesterone to synchronize oestrus and ovulation in cattle was first reported in 1960 (Hansel & Malven) and there is a considerable literature on the subject. For examp le, Hansel et al. ( 1966) found that 75% of treated anima ls were in oestrus by the end of the third day after MAP withdrawal. The remainder ca me into oestrus during the fourth, fifth and sixth days. A simi lar rate of return to oestrus using PIVS was reported by Ayalon & Marcus ( 1975) . Although this synchronization of oestrus is perhaps not optimal, it was still sufficient for all the non-pregnant cows in the present experiment to display, within three to four days after MAP withdrawa l, the low progesterone characteristic of oestrus so that accurate pregnancy determination could be made. This synchronization played a major role in the elimination of the 20% false positive rate associated with early pregnancy diagnosis based on milk progesterone.
PROGESTERONE AND PREGNANCY DETERMINATION
47
The use of PIVS, therefore, raises some interesting possibilities for the future improvement of early pregnancy diagnosis . If it is accepted that the normal rate of return to oestrus with PIVS is 75% within three days, then within 24 h following the removal of MAP, a very high percentage (at least 90%) of the herd cou ld be correct ly diagnosed since the drop in progesterone precedes oestrus by about 48 h . This would a ll ow the pregnancy diagnosis to be made in time for re-insemination and wou ld save the 20 open days lost when the diagnosis is availab le only after oestrus has a lready occurred. Effect of PI VS on fertility rate In the present study, a modest improvement in fertility was observed in cows which received exogenous progesterone in the post-insemination period as shown a lso by Wiltbank et al. ( 1956) and Johnson, Ross & Fourt ( 1958). This may be related to the observation that progesterone is essentia l for embryo survival in the cow (Hawk et al., 1963). The cattle in the present study normally had a 20% loss of embryos in the first 16 days following insemination (Aya lon, 1978); MAP treatment may prevent some of this. However, progesterone has not been widely used in commercia l breeding, perhaps due to reports sim il ar to that of Hansel et. al. ( 1966) that exogenous progesterone in the period prior to insemination reduces fertility by 10 to 15%. (It is of interest to note in this regard that three of the nine cows in the sub-ferti le herd, which had received progesterone in the pre-insemination period, became pregnant on the second insemination while none of the untreated cows conceived.) Of interest also is the improvement in fertility rate in the herd which consumed feed with high concentrations of phyto-oestrogens. It has been shown recently (Kaplansk i et. al., 1981) that one of the major effects ofphyto-oestrogens in cattle is the reduction of progesterone produced by the granulosa cells. It therefore seems logical that the exogeno us progesterone could have overcome this deficiency.
ACKNOWLEDGEMENT
This research was supported by Grant no. I-1-80 from the United States-Israel Binational Agricultural Research & Development Fund-BARD. REFERENCES
AVALON, N. ( 1978). Joumal of Reproduction and Fertility 54, 483. AYALON, N. & MARCUS, S. (1975). Theriogenology 3, 95. BALL, P. J. H. & POPE, G. S. ( 1976). Joumal of E11docrinology 69, 409. DARLING, j. A. B., LAING, j. A. & HARKNESS, R. A. ( 1974). joumal of Endocrinology 62, 29 1. DoBSON, H., MIOMER, S. E. & FITZPATRICK, R.j. (1975). Veterinary Record96, 222 . DONALDSON, L. E., BASSETr,j. M. & THORBURN, G . D. ( 1970).joumal of E11docrinology 48, 599. ERB , R. E., GAVERICK, H. A., RANDAL, R. D., BROWN, B. L. & CALLAHAN, C. J. (1976). Theriogenology 5, 227. HANSEL, W. & MALVERN, P. V. (1960) .Joumal of Animal Science 19, 1324. HANSEL, W., DONALDSON, E. L., WAGNER, W . C. & BRUNNER, M.A. ( 1966) .}oumal ofAnimal Science 25, 497. HANSEL, W., SCHECHTER, R.j ., MALVEN, P. V., SIMMONS, K. R., BLACK, D. L., HACKEIT, A.j. & S!\ATMAN, R. R. ( 1975) . Joumal of Animal Science 40,671.
48
BRITISH VETER INARY JOURNAL, 139, I
HAWK, H . W., BRINSFIELD, T. H., TURNER, G. D., WHITMORE, G. E. & NORCOSS, M.A. ( 1963) . J ournal of Dairy Science 46, 1397. H EAP, R. B., GwYN, M., LAING, .J. A. & \<\fALTERS, D. E. ( 1973). J ourna l of Agricultural Science, Cambridge 81, 151. HENDRICKS, D. M ., LAMOND, D. R., HII.L,j. R. & DICKEY,.]. F. (197 1).journalofAnimaiSciwce 33, 450 . HOFFMAN, B. & HAMBURGER, R. ( 1973). Zuc!ttlt)•giene 8, 154. HOFFMAN, B., HAMBURGER, R. , GONZLER, 0., KORNDORFER, L. & LOiiOFF, H . ( 1974). Theriogmology 2, 2 1. j OHNSON, K. R., Ross, R. H. & FOURT, B. L. ( 1958) . j ournal of Animal Sciwce 17, 386. KAPLANSKI, 0., SHt.MESH, M. & Bt.RMAN, A. ( 198 1). Journal of Endocrinology 89, 343. LAING,.J. A. & HEAP, R. B. ( 1971). British Veterina1J•J ournall27, xix. Port., G . S., MAJZI.IK, 1. , BALL, P.J. & LEAVER,.]. D. (1969) . Proceedingsoft!te8t!tlntemational Congress of Animal Reproduction and AI 3, I 72. Ronr.RTSON, H. A. ( 1972). Canadia n j ournal of Animal Science 52, 645. ROB INSON, T.j. ( 1965). Nature, London 206, 39. SHEM ESH, M ., AYALON, N. & LINDNER, H. R. ( 1968).journal of Reproduction and Fertility 15, 16 1. SHEMF.SH, M ., LINDNER, H . R. & AYALON, N. ( 1971 ). J oumal of ReJnoduction and Fertility 26, 127. SHEMESH, M., LINDNER, H. R. & AYALON, N. ( 1973). J oumal of ReJnoduction and Fertility 29, I. SHEMESH, M., AYALON, N. & MAZOR, T. ( 1979). j oumal of Reproduction and Fertilil)• 56, 30 1. SHEMESH, M., AYALON, N., MARCUS, S., DA NIELL!, Y., SHORE, L. & L.I\VI, S. (198 1). Theriogwology 15, 459. SHEMESH, M., AYALON, N., SHAI.EV, E., NERYA, A., SCHINDLER, H. & MILEGUIR, f. ( 1978) . Theriogenology 9, 343. STABENFELDT, G. H ., EWING, L. L. & McDONALD, L. E. ( 1969) . J ournal of Reproduction and Fertility 19, 433. THIDIER, M. (1974). Eliuage Insemination 144, 27. WILTBANK,.J. N., H AWK, H . W., KIDDER, H . E. E., UI.flERG, L. C. & CAS IDt\ , L. E. ( 1956) . J ournal of Dail)• Scimce 39, 456.
A NE\,Y APPROACH TO THE USE OF PROGESTERONE LEVELS FOR PREGNANCY DETERMINATION
BY M . SHEMESH, N. AVALON, S . LA VI, F. Mll.ECU IR , L. S. SHORE AND D . T OBY
Departments of Hormone Research and Reproduction, Ki1nron Veterinary Institute, Beil Dagan, P.O.B. 12, Israel
SUMMARY Intravaginal sponges impregna ted with 250 m g m ed roxyprogeste ro ne (MA P ) were in serted in Israe li Friesian cows a nd th e effect o n pl as m a progesterone a nd ea rly pregnancy di ag nosis based on milk progeste ron e o bse rved . It was found that:( I ) exogeno us MAP prevented the formati o n of a norm a l corpu s luteum if it was a dmini s tered for a number of d ays starting o n the d ay of oestrus. (2) In contrast, exoge no us MAP did not interfere with the d eve lopme nt of a normal corpus lute um wh en it was applied six or more d ays after oestrus. (3) R e m ova l of the MAP a fter the 17th d ay following oestrus resulted in a rapid fa ll in pl as m a a nd milk progesterone in non- pregna nt cows which (4) a ll owed a compl ete ly accurate determination o f pregna ncy to be m ade, e limin ati ng the 20% fa lse posi tives which us ua lly occur. (5) Fertility was not a dverse ly a ffected and (6) m ay act ua lly have been improved, es pecially in herd s with low fertility due to hig h con centra tion s of phyto-oest rogens in the feed .
INTRODUCT ION The d ay-to-d a y vanat1ons in pl as m a progesteron e in non-pregna nt cows display a cyclic p a ttern (Fig. I) ch aracterized by (a) minimal plasm a progeste ro n e levels, <0·5 ng/ml , fro m two d ays before to three days after ovulation, ( b) a sh a rp rise fro m day 5 to day 7 of th e cycl e, and (c) a very slow rise or platea u o n the s ubsequ ent eight to I 0 days, Ou c tu a ting about a m ean of 4 to 5 ng/ml. An importa nt fea ture of the bovine cycle is the abrupt fa ll in periphera l plasma progestero ne levels which occurs be tween th e third and second day before oes trus. Since thi s fall is not seen in pregna nt a nimals , an ea rl y pregn ancy diagnosis can be made at this time by progesterone determinations in plasm a (S hemesh, Ayalon & Lindn er, 1968; Pope et al., 1969; Sta benfeldt, Ewing & McDo na ld , 1969; Donaldson, Basse tt & Thorburn , 1970; Robertson, 1972) as well as in milk (Laing & H ea p, 1971; H ea p et al., 1973 ; Hoffm a n & Hamburger, 1973; Darling, Laing & Harkn ess, 1974;
BRITISH VETERINARY JO U RNA L, 139, I
42
Hoffm an et al., 1974; Thibier, 1974; Dobson, Midmer & Fitzpatrick, 1975; Ball & Pope, 1976; Shemesh et al., 1978). It is possible to use milk progesterone determin a tions since milk progesteron e accurately refle cts the pl as ma co ncentration of progeste rone (T ab le I).
7
6-
3-
{Oest r us)
Days after oestrus
Fig. I. Periph era l plasma progesterone levels in cows during the oes trous cycle (TLC/C PB meth od) . Cycle length 20 to 22 days, vert ica l bars indicate ± S£ (from Shemesh, Lindn er & A)•a lon, 197 1).
TABLE !
PROGEST ERON E CONCENTRAT IO N I N FORE M ILK AND JUGULAR PLASMA IN PREGNANT AND NON-PREGNANT CO WS AT 24 DAYS AFTER I NSEMINAT ION ( FROM SHEMESH ETAL., 1978)
Foremilk unextracted
Proges terone ng/ ml ± SEM n
Foremilk fat free
jugular plasma
Pregnant
Not pregnant
Pregnant
Not pregnant
Pregnant
Not pregnant
4· 73 ± 2·2 44
0·1-0·6 41
2·68 ± 1·8 21
0·1 29
2·9 ± 0·9 21
0·1-0·3 12
43
PROGESTERONE AND PREG NANCY DETERMINATION TABLE II
ACCURACY OF PREGNANCY DIAGNOSIS BY MILK PROGESTERONE AT 24 DAYS AFTER INSEMINATION (F ROM SHEMESH ET AL., 1978)
II
85
Diagnosed correctly pregnant
Diagnosed correctly not pregnant
No .
%
No.
%
34/44
78
41/41
100
Diagnosis of non-pregnancy can be routinely performed with 95 to 100% accuracy. However, when making a positive diagnosis only 70 to 80% accuracy has be en achieved (Table II); this error may be due to either lutea l phase progesteron e or early embryonic mortality. In an attempt to improve the accuracy of the test, progestinimpregnated vaginal sponges (PIVS) were applied to reduce the widespread range in the time of return to oestrus seen in insemin a ted cows which are not pregnant (Shemesh et. al. , 1981). The efficacy of the PIVS in inducing oestrus has been previously described (Ayalon & Marcus, 1975). Furthermore, in order to elucidate the mode of act ion of this exogenous progesterone, the early effect of PIVS on periph era l plasma progesterone at different periods of the oestrous cycle and following insemination was also st udi ed .
MATER IALS AND METHODS
Animals Experiment 1. Israeli Friesian heifers from a well-managed commercial herd were
housed in our Institute for three months. Animals were synchronized with Estrumate a nd on the day of oestrus, PIVS impregnated with 250 mg medroxyprogesterone (MAP) were inserted in five cows and withdrawn after 10 days. Jugular venous samples were taken a day before the ex pected oestrus (36 h after Estrumate) and on each of the following 12 days. Experiment 2. Nine heifers were inseminated and PIVS impregnated with MAP were inserted on day 6 after insemination (p.i.) and withdrawn on day 17.Jug ul ar venous blood samples were taken on day 6 p.i . and on each of the subseq uent 17 days. Experiment 3. The cows were Israeli Friesian cattle in well-managed commercial herds from the centra l plains area. Oestrus was detected by twice-daily observation. Six to seven days following routine artificial insemination animals either had PIVS inserted or were left untreated. The sponges were removed on day 17 p.i . Milk sa mples were taken 21 to 24 days after oestrus for pregnancy determinations. Experiment 1. Twenty-four dairy cows were used from a herd with poor fertility associated with high levels ofphyto-oestrogen. Thirteen cows had PIVS impregnated with MAP inserted and eleven served as controls . Animals which did not become pregnant following the first insemination were inseminated a second time.
44
BRI T I SH V ETERIN A RY J OU RNA L , 139, I
Measurement of plasma and milk progesterone concentrations T he m easu re ment of pl as ma a nd milk proges te rone conce ntrations was perform ed as previo us ly d escribed (Sh em es h , Aya lon & M azo r, 1979). C lini ca l co nfirm at ion of pregna ncy was based on recta l pa lpa ti on six to seven weeks post in se min a ti on .
RESU LTS
Experiment I. Exoge no us M A P in se rted imra vagin a ll y on th e d a y of oes tru s res ulted in non-d e tec table va lu es of proges tero ne throug hou t th e period studi ed ( <0· 1 ng/ml ). Th e M AP a ppa rentl y d id not cross- react with proges terone wh en th e specifi c a nti bod y aga in st progesterone- I Ia hemi succin a te in the presen t stud y was used . Exj;eriment 2. In se rtio n of M A P on d ay 6 aft er oes tru s, when th e co rpu s lute u m was a lread y es ta blished, did not a ffec t th e lutea l ph ase progeste rone in eith er p reg na nt (n =6) or non- pregna nt (n =3) heifers (Fi g. 2). Th e level of pl as ma proges terone in th e pregna n t heife rs was slightl y hi gh er th a n th a t of th e non-preg na nt heifers. H owever, within 24 h a fte r MAP withdra wa l, a ll three non-preg na nt heifers displayed a
.....__.... Pregnant
0 --{)
Non- pre gnant
Intra -vag inal
MAP
E
.....
(n =6l
"'c: c: e"'
~ V>
"'0 "'
a.
0
E
V>
0
a:
Days
fi g . 2. Th e effect o f intravagin a l s po nges impregna ted \vith M A P o n periph e ra l pl as ma proges tero ne level s wh en in se rted on d ay six a ft e r in semin a ti o n.
PROGESTERONE AND PREGNANCY DETERMINATION
45
dramatic decline in their plasma progesterone conce ntrations (from 4 to~ · 5 ng/ml) . Furthermore, within 62 h the plasma progesterone was less than 0·5 ng/ml and all of the heifers returned to oestrus . Experiment 3. The results of the laboratory diagnosis, co nfirmed by rectal palpation, are presented in Table II I. The accuracy of the test for the absence of pregnancy in cows 21 to 24 days after oestrus was I 00%, regardless of wheth er PIVS were used or not. However, markedly different results were found in positive pregnancy diagnosis. In co ntras t to the control group, in which 11 of the 52 {21%) cows were incorrec tly diagnosed as pregnant, all 30 cows with PIVS were correctly diagnosed as pregnan t. The fertility rate in the control group was 43% a nd in the PIVS group it was 60%. This indicates a possible beneficial effect on fertility .
TAI3LEIIJ ACCURACY Or PREGNAN CY DIAGNOSIS BY MILK PROGESTERONE AT 21 TO 24 DAYS AFTER OESTRUS WITH AND WITHOUT THE USE Or PIVS. PREGNANCY WAS CONFIRMED AT SIX TO SEVEN \'\'EE KS BY RECTAL PALPATION (FROM SHEMESH ETA!.. , 1981)
Treatment
No. of cows
Diagnosed correctly fmgnant
Diagnosed correctly not pregnant
No.
%
No.
%
79 100
44/44 20/20
100 100
None
96
41/52
PIVS
50
30/30
TABLE IV INFLUENCE OF MAP ON CONCEPTION RATE IN A DAIRY HERD \-\1 1TH POOR FERTILITY ASSOCIATED WITH HIGH LEVELS OF PHYTO-OESTROGENS. PREGNANCY WAS CONFIRMED AT SIX TO SEVEN WEEKS BY RECTA L PALPATIO
Treatment
Conception rate
No. of cows 1st insemination
None
11
PIVS
13
1
2nd insemination
Total
No .
%
No .
%
No.
2/11 4/13
18
31
0/9 3/9
33
2/11 7/13
18 54
Experiment 4. The rate of conception in these sub-fertile cows with or without PIVS is shown in Table IV . The cows with PIVS had a 54% conception rate compared with 18% in the controls. This herd had clinical indications- high incidence of follicular cysts, nymphomania and short intervals between oestrous periods - that the infertility was due to high leve ls ofphyto-oestrogens in the feed . In the laboratory, it
46
BRITISH VETERINARYJOURNAL, 139, I
was found that the level of phyto-oestrogens in the various feed constituents ranged from the equivalent of 61 to 448 ng oestradiol/g dry matter as determined by radioreceptor assay (Shemesh, Lindner & Ayalon, 1973) and in-vitro bioassay (Kaplanski, Shemesh & Berman, 1981). DISCUSSION
Value of progesterone determinations in milk and plasmafor pregnancy detennination The determination of plasma progesterone gives an accurate picture of the status of the corpus luteum in the cow, i.e., following luteolysis, plasma progesterone falls rapidly to non-detectable levels. Defatted milk also accurately reflects the plasma concentration of progesterone. This study shows that l 00% correct pregn a ncy diagnosi s can be obtained if exogenous progesterone (MAP) is administered during the post-insemination period. Effect of exogenous MAP on plasma progesterone concentrations Exogenous MAP given on the day prior to expected oestrus prevented the form at ion of the corpus luteum as reflected by the absence oflutea l progesterone. The underlying mechanism involved is most probably connected with the fact that progesterone acts to suppress pituitary LH release in th e non-pregnant cow (Hansel et al., 1975 ). However, once the corpus luteum was established, MAP did not affect the plasm a progesterone concentration. The MAP did not therefore interfere with the functional corpus luteum. Indeed , the slightly higher proges terone concentrations associated with corpora lutea in the presence of a conceptus which have been reported by others (Hendricks et al., 1971; Erb et al., 1976; H anse l & Lukaszewska, personal communication) were seen in this study even when MAP was present. Effect of PI VS on improving the accuracy of milk progesterone for early pregnancy diagnosis In 1965, Robinson reported a new approach to oestrus synchronization in sheep using vaginal sponges to which a progestin compound was adsorbed. The method is widely used for sheep but in cattle it has not been commercially successful since experiments have not given uniform resu lts and many catt le failed to retain the sponge. However, Ayalon & Marcus ( 1975) have obtained l 00% retention rates using a locally-produced intravaginal sponge. The use of exogenous progesterone to synchronize oestrus and ovulation in cattle was first reported in 1960 (Hansel & Malven) and there is a considerable literature on the subject. For examp le, Hansel et al. ( 1966) found that 75% of treated anima ls were in oestrus by the end of the third day after MAP withdrawal. The remainder ca me into oestrus during the fourth, fifth and sixth days. A simi lar rate of return to oestrus using PIVS was reported by Ayalon & Marcus ( 1975) . Although this synchronization of oestrus is perhaps not optimal, it was still sufficient for all the non-pregnant cows in the present experiment to display, within three to four days after MAP withdrawa l, the low progesterone characteristic of oestrus so that accurate pregnancy determination could be made. This synchronization played a major role in the elimination of the 20% false positive rate associated with early pregnancy diagnosis based on milk progesterone.
PROGESTERONE AND PREGNANCY DETERMINATION
47
The use of PIVS, therefore, raises some interesting possibilities for the future improvement of early pregnancy diagnosis . If it is accepted that the normal rate of return to oestrus with PIVS is 75% within three days, then within 24 h following the removal of MAP, a very high percentage (at least 90%) of the herd cou ld be correct ly diagnosed since the drop in progesterone precedes oestrus by about 48 h . This would a ll ow the pregnancy diagnosis to be made in time for re-insemination and wou ld save the 20 open days lost when the diagnosis is availab le only after oestrus has a lready occurred. Effect of PI VS on fertility rate In the present study, a modest improvement in fertility was observed in cows which received exogenous progesterone in the post-insemination period as shown a lso by Wiltbank et al. ( 1956) and Johnson, Ross & Fourt ( 1958). This may be related to the observation that progesterone is essentia l for embryo survival in the cow (Hawk et al., 1963). The cattle in the present study normally had a 20% loss of embryos in the first 16 days following insemination (Aya lon, 1978); MAP treatment may prevent some of this. However, progesterone has not been widely used in commercia l breeding, perhaps due to reports sim il ar to that of Hansel et. al. ( 1966) that exogenous progesterone in the period prior to insemination reduces fertility by 10 to 15%. (It is of interest to note in this regard that three of the nine cows in the sub-ferti le herd, which had received progesterone in the pre-insemination period, became pregnant on the second insemination while none of the untreated cows conceived.) Of interest also is the improvement in fertility rate in the herd which consumed feed with high concentrations of phyto-oestrogens. It has been shown recently (Kaplansk i et. al., 1981) that one of the major effects ofphyto-oestrogens in cattle is the reduction of progesterone produced by the granulosa cells. It therefore seems logical that the exogeno us progesterone could have overcome this deficiency.
ACKNOWLEDGEMENT
This research was supported by Grant no. I-1-80 from the United States-Israel Binational Agricultural Research & Development Fund-BARD. REFERENCES
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BRITISH VETER INARY JOURNAL, 139, I
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