Animal Reproduction Science, 4 (1981) 93--106 93 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

C O N C E N T R A T I O N S O F O E S T R A D I O L - 1 7 / 3 A N D P R O G E S T E R O N E IN

T H E P L A S M A O F D A I R Y H E I F E R S B E F O R E A N D A F T E R

C L O P R O S T E N O L - I N D U C E D A N D N A T U R A L L U T E O L Y S I S A N D

D U R I N G E A R L Y P R E G N A N C Y

R.G. GLENCROSS and G.S. POPE

Department of Physiology, National Institute for Research in Dairying, Shinfield, Reading, Berks, RG2 9AT (Great Britain)

(Accepted 11 May 1981)

ABSTRACT

Glencross, R.G. and Pope, G.S., 1981. Concentrations of oestradiol-17~ and progesterone in the plasma of dairy heifers before and after cloprostenol-induced and natural luteolysis and during early pregnancy. Anita. Reprod. Sci., 4 :93 -106.

Plasma oestradiol-17~ and progesterone levels were measured in seven nulliparous, dairy heifers (British Friesian breed) that were administered cloprostenol (a synthetic analogue of prostaglandin F 2 a) between days 8 and 14 of the oestrous cycle and inseminated (AI) 72 and 96 h later, and in seven heifers inseminated (AI) at natural oestrus.

In both treated and untreated heifers, the beginning of the progesterone fall and the oestradiol-17~ rise associated with luteolysis appeared to be synchronous but, whereas the rate of fall in progesterone level was greater for the treated heifers, that of the oestra- diol-17~ rise did not differ between treated and untreated heifers. Mean pre-ovulatory peaks of oestradiol-17~ were 8 pg/ml and 10 pg/ml for treated and untreated heifers re- spectively.

A post-ovulatory peak of oestradiol-17~ in plasma 5--6 days after the pre-ovulatory peak occurred in all heifers whether or not conception had taken place. It is suggested that 7 days after the initiation of oestradiol-17~ secretion by the pre-ovulatory follicle, another follicle begins to mature and secrete oestradiol-17~ and that the progress of the latter towards full maturation and potential ovulation is stopped by rising progesterone levels from the corpus luteum; as a result in normal, non-pregnant cattle an interval of about 21 days elapses before another ovulation (of another follicle) takes place. In the event of premature luteolysis (in the present study induced between the 8th and 14th day) there is no evidence that the timing of this luteolysis influences the time taken for a fol- licle to enter the final stages of pre-ovulatory maturation, when increasing amounts of oestradiol-17fl are secreted. Thus the interval between ovulations may not be less than 7 days but, depending on corpus luteum survival, may vary between 7 and 21 days.

In one heifer after natural luteolysis a normal plasma oestradiol-17fl peak followed but this was not associated with ovulation and corpus luteum formation. The second oestradiol- 17~ peak 6 days after the first, however, evidently assumed the ovulatory role; presum- ably the secreting follicle concerned, not being subject to inhibition by progesterone ris- ing to luteal levels, matured fully and ovulated. Thus the second, normally post-ovulatory, oestradiol-17~ peak in cattle can, in the event of failure of ovulation at the normal time, itself assume the ovulatory function, the oestrous cycle length then being about 28 days.

0378-4320/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company

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INTRODUCTION

Rowson et al. {1972) showed that intrauterine administration of prosta- glandin F2a (PGF~a) to cattle between days 5 and 16 of the oestrous cycle caused premature regression of the corpus luteum (luteolysis) and resulted in oestrus 3 days later. Intramuscular injection of cloprostenol (a synthetic analogue of PGF2a) was also luteolytic in heifers when given at these times (Tervit et al., 1973), and Cooper (1974) reported that its use provided a meth- od for synchronization of oestrus in herds and offered the possibility of suc- cessful artificial insemination (AI) at pre-determined times.

This possibility was investigated by observing calving rates in dairy heifers that were administered cloprostenol and inseminated 72 h and 96 h {or 72 h only) after treatment (Leaver et al., 1975). As part of this investigation, levels in plasma of oestradiol-17~ and progesterone were also measured to compare the timing of follicular maturation and ovulation after cloprostenol- induced and natural luteolysis. The results of this latter part of the study are now reported.

Although there is general agreement on the concentrations of plasma progesterone in cattle, the reported levels of plasma oestradiol-17/~ differ considerably (Smith et al., 1975, 1979; Peterson et al., 1975; Chenault et al., 1976; Chung, 1979). Because of this, special attention has been given to validation of the assay for oestradiol-17g.

MATERIALS

Materials and apparatus used were as described by Glencross et al. (1973) with the following exceptions.

Steroids

[1,2,6,7-3H]progesterone (82 Ci/mmol), code no. TRK 413, was purchased from the Radiochemical Centre (Amersham, Bucks, United Kingdom).

Oestradiol-17a (lot no. 47301) was purchased from Koch-Light Laborato- ries Ltd. (Colnbrook, Bucks, United Kingdom).

Chromatography

Chromatographic adsorption grade silica gel (60--120 mesh) was purchased from BDH Chemicals Ltd (Pool% Dorset, United Kingdom). 0.5 g was retained in a 20-ml capacity glass column (15 cm X 1.5 cm), which had a ground glass cone at the lower end, by means of a loose-fitting, cylindrical glass plug.

For Sephadex LH-20 gel partition chromatography (Pharmacia, GB, Ltd, Hounslow, Middlesex, United Kingdom) a column of dimensions 9 cm X 0.7 cm was used, the material being held in place with a glass-wool plug.

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Antisera

Antisera A, C and E (Glencross et al., 1973) were used. Antiserum A, which was raised in sheep against oestradiol-17~,17/~- succinyl, BSA (Thorneycroft et al., 1970), was used solely in tests of the specificity of the oestradiol-17~ assay. Antiserum C, raised against oestradiol-17~, 6(0-carboxymethyloxime), BSA (Dean et al., 1971) in rabbit (Exley et al., 1971) was used in the assay of the plasma samples for oestradiol-17~.

Progesterone was assayed using antiserum E, raised in goat against l l a - hydroxyprogesterone, succinyl, BSA (Furr, 1973).

Assay tubes and washing of glassware

Borosilicate glass tubes, 8.5 cm × 1.1 cm and 8.0 cm × 0.9 cm, were used for oestradiol-17/3 and progesterone radioimmunoassay respectively.

Glassware was machine-washed using a programme incorporating deionized, cold water rinses, hot alkaline detergent and acid washes and hot tap water and hot distilled water rinses.

Dextran-coated charcoal suspension

A suspension of 6.25 g radioimmunoassay grade charcoal (Becton, Dickin- son, UK, Ltd, Wembley, Middlesex, United Kingdom} with 0.625 g dextran 20 (lot no. TO 5401; Pharmacia, GB, Ltd) in 1 1 of assay buffer was used.

Radioactivity counting

Radioactivity of samples was counted Cat least 1000 cpm) using a Liquid Scintillation Spectrometer, model PW 4510/01 (Philips, Eindhoven, The Netherlands) and a 226Radium external standard.

METHODS

Experimental heifers and plasma collection

Fourteen of the nulliparous, British Friesian heifers, described by Leaver et al. (1975} in their experiment 1, were used. Seven received a single, intra- muscular injection of 0.5 mg of the PGF2~ analogue, cloprostenol (ICI 80996; ICI Pharmaceuticals Divison, Macclesfield, Cheshire, United Kingdom) at a known time during the mid-luteal phase of the oestrous cycle and were in- seminated (AI) 72 h and 96 h later. The other seven were inseminated (AI) at naturally-occurring oestrus.

Jugular-vein blood (50 ml) was collected daily (09.00--10.00 h} for 32 days from all 14 heifers using heparinized, evacuated glass tubes. This period stretched from before cloprostenol-induced or natural luteolysis, until subse-

96

quent oestrus when AI was performed, and for some days later when most heifers were pregnant. Plasma was separated by the method of Glencross et al. (1973).

Additionally, single jugular-vein blood samples (0.2--0.5 1) were collected in heparinized, polyethylene bottles from another five untreated heifers 12-- 24 h after the end of observed oestrus, at a time when plasma oestradiol-17~ and progesterone concentrations are low (Glencross et al., 1981). Plasma from these samples, separated as above, was used in the validation of the steroid assay procedures.

Plasma oestradiol-17~ assay

The plasma samples (in duplicate) from a cloprostenol-treated and a control heifer were assayed together. Included in the same batch of assayed samples as quality controls were duplicate volumes of distilled water (10 ml) and seven oestradiol-17fl standards (0, 10, 20, 30, 50, 70 and 100 pg), each in duplicate, and to each of which pooled plasma (10 ml) low in endogenous oestradiol- 17/3 was added. All the samples, including those for quality control, were ex- tracted and assayed for oestradiol-17~ as follows.

[3H] oestradiol-17/3 (10 pg; 6600 dpm) in toluene (0.1 ml) was added to 100 ml conical glass flasks and the toluene evaporated. The plasma (or water) samples (10 ml) were added to the flasks and left for 17--20 h at 4°C in the dark, after which it was assumed that the tritiated and any endogenous oestradiol-17/3 were homogenous.

Oestradiol-17~ was extracted by shaking the sample with toluene (3 X 20 ml) at 20--23°C for 15 min on an orbital shaker (180 rev/min). Emulsions which formed were broken by repeated (two to three times) freezing (ethanol bath at --40°C; 5 min) and thawing (water bath at 70°C; 5 min). The toluene extracts were decanted from the frozen plasma. The three 20-ml volumes of toluene extract were poured successively through silica gel (0.5 g) chroma- tographic columns, the effluent being discarded. The oestradiol-17/3, adsorbed on the silica gel, was eluted with methanol (0.7 ml) directly onto a Sephadex LH-20 gel column (9 cm X 1 cm), from which it was eluted with methanol/ toluene (15 : 85 v/v). The first 2.9 ml eluate {measured from the addition of the methanol) was discarded and the next 3.5 ml collected for oestradiol-17~ estimation.

The silica gel, which was washed with methanol (10 ml) and toluene (20 ml) before re-use, was discarded after processing 12 extracts. The Sephadex LH-20 gel columns, which were washed with elution solvent (20 ml) before re-use, were used repeatedly.

Oestradiol-17~ was estimated in the plasma extract, after evaporation of the elution solvent, by the radioimmunoassay procedure of Glencross et al. {1973) with the following modifications.

Oestradiol-17~ standards (single quantities of 0, 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 25, 30, 45, 50, 60, 80, 100, 125 and 150 pg), [3H] oestradiol-17~

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(6 pg; 4000 dpm) and diluted antiserum C (1 : 250 000; 0.5 ml/tube) were used. Dextran-coated charcoal suspension (0.2 ml) was used to separate the free from antibody-bound steroid. The concentrations of oestradiol-17~ in the plasma samples were calculated using the cubic expression of best fit to the standard inhibition curve (ICL system 4-70 computer; Rothamsted Ex- perimental Station, Harpenden, Herts, United Kingdom).

Validation of the oestradiol-17~ assay

Reagent blank, sensitivity and precision. The mean estimates of oestradiol- 17~ in water (10 ml) that was included as a quality control in each of seven batches of plasma samples (reagent blank) was 9.5 + 1.3 pg~ oestradiol-17/3.

Sensitivity and precision were calculated from levels of oestradiol-17~ estimated in sub-samples (10 ml) of the plasma pool of low endogenous oestradiol-17/~ level, to which known quantities of standard oestradiol-17/3 were added and which were included in each of seven batches of plasma samples. Sensitivity of 20 pg oestradiol-17~, i.e. 2 pg/ml (Anastassiadis and Common, 1968), and within-assay and between-assay coefficient of varia- tion of 16% and 26% respectively (Rodbard, 1974), were determined {mean for 0--100 pg).

Accuracy. The accuracy of the plasma oestradiol-17/~ assay was assessed by adding 24 single quantities of oestradiol-17~ standards {0--50 pg in 5 pg in- crements and 60--180 pg in 10 pg increments) to sub-samples of the plasma pool of low endogenous oestradiol-17~ concentration and estimating the levels of oestradiol-17~. Regression analysis of estimated upon known values of added oestradiol-17/3 gave a linear relationship y = 23 + 1.06x (95% con- fidence limit of slope 0.95, 1.17).

Specificity. Solvent extraction of plasma oestrogens and purification by silica gel and Sephadex LH-20 chromatography were designed to separate and remove most plasma constituents, including some steroids, that might inter- fere in the assay. However, structurally-related oestrogens might still be present in the purified plasma extracts that could bind to the antisera and result in erroneous oestradiol-17~ estimations. Two such oestrogens, the levels of which have been measured in the plasma of cattle during the oestrous cycle, are oestrone and oestradiol-17a (Dobson and Dean, 1974; Seren et al., 1976). To investigate possible interference from these steroids in the assay of oestradiol-17~ in bovine plasma the following study was undertaken.

The two oestradiol-17~ antisera A and C, both of which were known to have little or no cross-reaction (0.1% or less} with steroids other than oestro- gens, but significant (and different) cross-reactions with other oestrogens (Exley et al., 1971; Glencross et al., 1973) were rigorously retested for their specificity of binding to oestrone and oestradiol-17a.

To three series of sub-samples (10 ml) of the same pooled plasma of low

i SEM given here an d in all s u b s e q u e n t m e a n values.

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endogenous oestradiol-17~ level, were added respectively, 12 increasing quantities of oestradiol-17/~ (0--160 pg), 11 of oestrone (0--250 pg) and 12 of oestradiol-17~ (0--1 ng). The added oestrogen was then estimated in equiv- alents of oestradiol-17~ by the radioimmunoassay method under validation using antiserum A (1 : 400 000 dilution). The procedure was repeated for an- other three series of similar plasma sub-samples, to which 12 increasing quan- tities of oestradiol-17/~ (0--160 pg), 12 of oestrone (0--37.5 ng) and 11 of oestradiol-17~ (0--100 ng) respectively had been added, using antiserum C (1 : 250 000 dilution}.

Regression analysis for all six series of estimated oestrogen (as oestradiol- 17~ equivalents} against added oestrogen gave linear results (Fig. 1). Cross- reactions of oestrone and oestradiol-17~ to each antiserum were then ex- pressed as the ratio of the regression coefficient (slope of line) of the oestro- gen to that of oestradiol-17~ added to plasma. A cross-reaction for oestrone of 50% and of oestradiol-17~ of 12% was calculated for antiserum A, and 0.6% for oestrone and 0.1% for oestradiol-17~ for antiserum C.

To investigate the possible presence of oestrone and oestradiol-17~ in the plasma of cattle during the time of the pre-ovulatory follicular peak of oestra- diol-17~ (Glencross et al., 1973, 1981}, single plasma samples (100--150 ml) were taken from four cows and two heifers 12--48 h before oestrus, i.e. dur- ing the pre-ovulatory period when plasma oestradiol-17~ levels are elevated. Sub-samples (10 ml) of the plasma samples were extracted, and assayed for oestradiol-17~, half of the samples from each animal with antiserum A (1 :

250

200

150

Oestradiol- 1713

Oestradiol-17~

Oestrone o o [J

Oestrone

' ' ' ' '6 ' 250 500 750 1 0 5 50 7 100

Steroid oestrogen added (Pg) Steroid oestrogen added (ng)

Fig. 1. Estimated quantities of oestradiol-17~ (circle), oestrone (triangle) and oestradiol- 17~ (square) (expressed as oestradiol-17~) that were added to plasma (10 ml), assayed us- ing antiserum A raised against a C-17 conjugate (open symbols), and antiserum C raised against a C-6 conjugate (closed symbols).

99

400 000 dilution) and the others with antiserum C (1 : 250 000 dilution). The concentrations of oestradiol-17~ estimated in these follicular phase

plasma samples using antiserum C {mean 8.1 pg/ml) were slightly lower than those using antiserum A (mean 9.1 pg/ml), the difference (2.1 pg/ml) for one heifer, and that for mean values (1.0 pg/ml) reaching significance at the 95% level.

For the latter difference (1.0 pg/ml) to be due either to oestrone or to oestradiol-17a, it was calculated, from the estimated cross-reactions, that the concentrations of oestrone or oestradiol-17a in the pre-ovulatory plasma needed to be as high as 2.0 pg/ml or 8.7 pg/ml respectively. Since the oestra- diol-17~ specific antiserum C was used for all plasma samples, other than those involved with the testing of specificity, the estimates of oestradiol-17~ in plasma taken at the time of the pre-ovulatory peak are unlikely to be in error by more than 0.02 pg/ml due to the presence of oestrone or oestradiol- 17a, a negligible error far below the sensitivity of the assay.

Plasma progesterone assay

Each assay (i.e. batch of samples assayed together) included all the plasma samples in duplicate from one heifer. Also submitted to each assay to serve as quality controls were duplicate samples of distilled water (0.2 ml) and luteal phase plasma (0.2 ml), known to have high progesterone level.

Plasma progesterone was measured by the method of Glencross et al. (1973) with the following modifications.

Plasma (or water) samples (0.2 ml), to which [3H] progesterone (9 pg; 5000 dpm) was added, were extracted with light petroleum, b.p. 40--60° C (2 × 2.5 ml). Progesterone in the extract was measured using progesterone standards (0, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 ng), in duplicate and [3H]pro- gesterone (7 pg; 4000 dpm) in the standard tubes and using diluted anti- serum E (1 : 15 000; 0.5 ml/tube).

Validation of the progesterone assay

Reagent blank and precision. Water and sub-samples of plasma pools were included as quality control samples in all 14 batches of samples assayed.

From estimates for the duplicate samples of water, a reagent blank of 56 + 8 pg was obtained. From the estimated levels of progesterone in the luteal phase plasma (5.6 -+ 0.2 ng/ml), the intra-assay and inter-assay coeffi- cients of variation were calculated to be 4% and 9% respectively.

Sensitivity and accuracy. Overall sensitivity and accuracy of the assay was examined by adding progesterone standards (0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.4 and 3.2 ng), each replicated six times, to sub-samples of plasma of low endogenous progesterone level and measuring progesterone content.

By the method used to validate the sensitivity and accuracy of the oestra-

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diol-17~ assay, an assay sensitivity of 15 pg progesterone, i.e. 0.08 ng/ml, and a regression equation (estimated upon known added values of progesterone) y = 0.1 + 1.03x (95% confidence limit of slope 1.08, 1.17) was obtained.

Specificity. To test specificity, light petroleum extracts of plasma samples (0.5 ml) taken from a heifer during oestrus, a heifer in the luteal phase of the oestrous cycle and a pregnant heifer, to which [3H] progesterone had been added, were chromatographed on a Sephadex (LH-20) column (40 cm X 1 cm) in 80 : 20 v/v, cyclohexane/ethanol (Setchell and Shackleton, 1973). The first 30 fractions (3 ml each) were collected. Only fractions containing pro- gesterone, as estimated by the radioimmunoassay (0.05, 4.45 and 8.35 ng/ml plasma respectively for samples taken at oestrus, in the luteal phase of the cycle and in pregnancy) had peak levels of radioactivity.

RESULTS

Luteolysis was induced in all seven heifers to which cloprostenol was administered between days 8 to 14 of the oestrous cycle. Two of the heifers showed oestrus 3 days later, four on the 4th day and one on the 7th day after treatment.

As shown in Fig. 2, plasma progesterone levels were at luteal levels in all heifers before the administration of cloprostenol indicating that ovulation and corpus luteum development had occurred after the previously observed oestrus. In the four heifers whose plasma oestradiol-17~ level was measured early in this luteal phase, a peak of 4.8 + 0.4 pg/ml was found around day 5 to 6 of the cycle when the plasma progesterone level was 1.8 + 0.2 ng/ml. At the time of cloprostenol treatment, oestradiol-17/3 in the plasma of all seven heifers was at basal level. After cloprostenol administration, the level of plasma progesterone fell from an average concentration of 6.2 + 0.7 ng/ml to 0.6 + 0.1 ng/ml within 24 h and had reached basal level of 0.3 + 0.1 ng/ml by the next day. With the exception of heifer 967, plasma oestradiol-17~ levels rose during this period of luteal regression from a mean basal concen- tration of 1.0 + 0.2 pg/ml on the day of cloprostenol t reatment to 3.5 +- 0.7 pg/ml by the following day and to 5.6 + 1.1 pg/ml by the next. (In heifer 967, this rise in plasma oestradiol-17~ was delayed for 2 days.) During the period of 5 to 9 days when plasma progesterone remained at a basal level of around 0.1--0.3 ng/ml, oestradiol-17~ reached a peak level of 8.4 + 0.6 pg/ml for a 1- to 2-day period before declining rapidly to a basal level of 1.2 + 0.3 pg/ml within 24 h. During this period oestrus was exhibited and the seven heifers were inseminated (AI); six became pregnant. Plasma progesterone levels rose slowly from basal levels 2 days after oestrus to 5--6 ng/ml during the following 9 to 10 days. Plasma oestradiol-17~ levels, also low for 2 to 3 days following oestrus, also increased for a 3- to 4-day period after oestrus. The post-ovulatory peak in plasma oestradiol-17~ concentrat ion (4.9 -+ 0.8 pg/ml) was found on the 4th or 5th day after AI in all six pregnant and the

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Fig. 2. Concentrations of plasma oestradio]-I 7B and progesterone in heifers that were administered cloprostenol between day 8--14, C (n), o f the oestrous cycle and were in- seminated (AI) 72 h and 96 h later and (for heifer 967) at observed oestrus.

one non-pregnant heifer, at a time when plasma progesterone levels had risen to 1.5 + 0.1 ng/ml.

Of the seven untreated heifers, six showed oestrus after a normal 20 to 21 day cycle, whereas the seventh showed oestrus after an abnormally long (28-day) cycle (Fig. 3). For the six heifers for which complete data were available (i.e. excluding heifer 902; Fig. 3), the fall in plasma progesterone as- sociated with natural luteolysis was from a mean concentration of 5.5 -+ 0.6

102

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Fig. 3. Concentrations of plasma oestradiol-17~ and progesterone during natural luteolysis and in early pregnancy in heifers inseminated (AI) at observed oestrus.

ng/ml to a mean of 2.8 + 0.5 ng/ml within 24 h and to 0.6 + 0.1 ng/ml after 48 h. During this time, plasma oestradiol-17~, which was at a low level of 2.2 + 0.5 pg/ml at the start of luteal regression, had increased to 3.8 + 0.6 pg/ml by the following day, when plasma progesterone levels were still elevated (2.8 + 0.5 ng/ml) and had reached 6.6 + 0.9 pg/ml by the day plasma pro- gesterone levels were basal. In the six heifers where plasma progesterone levels remained basal for 5 to 6 days, plasma oestradiol-17fl levels showed a peak of 10.1 + 0.5 pg/ml, 1 or 2 days after complete luteolysis, before returning rapidly to basal values of 1.4 + 0.2 pg/ml by the following day. All six heifers showed oestrus around this time and became pregnant to insemination (AI}.

The seventh heifer, 992, although showing an oestradiol-17~ peak of 8 - 9 pg/ml, did not show oestrus and, from the lack of elevated progesterone levels in the following 6 to 7 days, presumably did not ovulate either. Only after another smaller oestradiol-17~ peak of about 6 pg/ml 6 days later, was oestrus observed. Insemination at this oestrus resulted in pregnancy.

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Following oestrus and AI, the plasma progesterone and oestradiol-17~ levels in the six untreated heifers measured during early pregnancy showed a similar pattern to those found in the pregnant cloprostenol-treated heifers. The second (post-ovulatory) oestradiol-17/~ peak which occurred on day 5 of pregnancy reached a mean concentration of 4.7 + 0.5 pg/ml when plasma progesterone had reached 1.8 -+ 0.2 ng/ml.

DISCUSSION

From the estimated levels of oestradiol-17~ in plasma samples taken at oestrus, using two antisera having different cross-reactions to oestrone and oestradiol-17a, it is concluded that, whereas there two oestrogens may have been present in plasma at the time of the pre-ovulatory peak of oestradiol- 17/~ in measurable concentrations, these were not high enough to influence significantly the estimate of plasma oestradiol-17/~ concentration obtained at this time with the highly specific antiserum C. The estimated possible maxi- mum levels of 2 pg/ml oestrone and 9 l~g/ml oestradiol-17a in plasma at the time of the pre-ovulatory oestradiol-17/~ peak are consistent with the range of estimates reported for plasma oestrone in cattle following natural luteolysis (Echternkamp and Hansel, 1973; Glencross et al., 1973; Dobson and Dean, 1974; Smith et al., 1975; Seren et al., 1976) and after PGF2~-induced luteo- lysis (Fogwell et al., 1978) and also for plasma oestradiol-17a (Dobson and Dean, 1974; Seren et al., 1976).

The changes in levels of plasma progesterone and oestradiol-17~, which show that cloprostenol administered to heifers in the mid-luteal phase of the oestrous cycle induces luteolysis and is followed by oestrus and ovulation 3--4 days later, are in agreement with those reported by Dobson et al. (1975) and Cooper and Rowson (1975). Similar results have also been reported in cattle after PGF2a administration (Louis et al., 1974; Henricks et al., 1974; Chenault et al., 1976; Thatcher and Chenault, 1976; Stellflug et al., 1977; Fogwell et al., 1978; Smith et al., 1979).

The evidence from six out of the seven treated heifers was that the stage of the luteal phase when premature luteolysis was effected had no influence on the interval from treatment to oestrus, suggesting that a follicle is normally ready to enter the pre-ovulatory state, with increasing secretion of oestradiol- 17~, at any time of the mid-luteal phase immediately the secretory function of the corpus luteum is removed.

During natural luteolysis plasma progesterone fell from luteal to basal levels in 48 h whereas after cloprostenol treatment a similar fall occurred in the first 24 h. However, the pattern of rise in plasma oestradiol-17/~ was es- sentially similar during and after natural and induced luteolysis, the rise begin- ning from the time of the first fall in plasma progesterone level and reaching a peak 3 to 4 days later. These results are consistent with the proposal that it is the first sharp fall in plasma progesterone associated with luteal regression which normally permits oestradiol-17~ secretion from the pre-ovulatory fol-

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licle to proceed (FogweU et al., 1978; Karsch et al., 1978). It may be noted, however, that this secretion continues at an increased rate at a time during natural luteolysis when progesterone levels, though falling rapidly, are still well above basal (Chow et al., 1974; Glencross et al., 1981).

A post-ovulatory peak in plasma oestradiol-17/~ levels in cattle around the 5th or 6th day after oestrus (also present when conception has taken place) has already been reported (Glencross et al., 1973; Smith et al., 1975). It is thought to be due to secretion from a follicle at this time whose maturation towards ovulation has begun but is later inhibited by the rising level of plasma progesterone. Thus there is an interval of about 7 days between the first rise in plasma oestradiol-17~ due to an ovulatory follicle and the first rise due to this presumed post-ovulatory follicle.

It appears likely, and is a subject for further study, that there is physiolog- ically a necessary minimum period of 7 days after the first rise in plasma oestradiol-17~ due to a pre-ovulatory follicle and a second such rise due to a potentially ovulatory follicle. Physiologically, in the normal non-pregnant cow this period is extended to 21 days due to the function of the corpus luteum but may be reduced to any length between 7 and 21 days if premature luteo- lysis is induced.

The peak oestradiol-17~ level, 5--6 days after oestrus, is similar in non- pregnant cattle and in cattle conceiving at this oestrus but whether it has any physiological function is unknown. Exceptional among the untreated heifers, however, is heifer 992 (Fig. 3) in which it appeared that, whereas the plasma oestradiol-17~ level rise at the time of natural luteolysis reached a peak of normal magnitude, the follicle concerned did not, according to subsequent progesterone levels, ovulate. However, the second oestradiol-17/3 peak, 6 days later, did induce oestrus and ovulation since the heifer, inseminated at this time, became pregnant. Thus the second oestradiol-17~ peak 9 days after the initiation of natural luteolysis and 6 days after the previous peak has in this case assumed the pre-ovulatory role following default of the normal pre- ovulatory peak in this respect.

Exceptional among the treated heifers was heifer 967, which showed the characteristic fall in plasma progesterone level due to induced luteolysis but not the accompanying rise in plasma oestradiol-17~ until 2 days after plasma progesterone had reached basal levels. The peak of oestradiol-17~ level in the following 5 days was otherwise quite normal, inducing oestrus and ovulation (the heifer was inseminated at this oestrus and became pregnant). This delay in the rise of plasma oestradiol-17/~ level following induced luteolysis was presumably due to delay in development of a follicle able to begin oestradiol- 17/3 synthesis (Scaramuzzi et al., 1980}. This prolongation of the period from treatment to ovulation offers some explanation for the long follicular phase that is sometimes apparent from progesterone levels after prostaglandin-in- duced luteolysis in cows and which prevents fixed-time AI succeeding (Baishya et al., 1980).

105

ACKNOWLEDGEMENTS

The authors wish to thank Dr J.D. Leaver, Mr N.H. Yarrow and their staff at the heifer unit, Arborfield Hall Farm, NIRD for their care of the experi- mental heifers and for their help in the collection of blood samples and with the administration of cloprostenol. The help of Mrs P.A. Sheffield with sam- ple collection and for the assay of progesterone is also very much appreciated.

The authors are also grateful to ICI Pharmaceuticals Division, Macclesfield for the gifts of cloprostenol and to Dr B.V. Caldwell, Dr D. Exley and Dr B.J.A. Furr for the gifts of antisera.

REFERENCES

Anastassiadis, P.A. and Common, R.H., 1968. Some aspects of the reliability of chemical analyses. Analyt. Bioehem., 22: 409--423.

Baishya, N., Ball, P.J.H., Leaver, J.D. and Pope, G.S., 1980. Fert i l i ty of lactating dairy cows inseminated after t reatment with cloprostenol. Br. Vet. J., 136: 227--239.

Chenault, J.R., Thatcher, W.W., Kalra, P.S., Abrams, R.M. and Wilcox, C.J., 1976. Plasma progestins, estradiol, and luteinizing hormone following prostaglandin F2~ injection. J. Dairy Sci., 59: 1342--1346.

Chow, L.A., Thatcher, W.W., Kalra, P.S. and Chenault, J.C., 1974. NIveis sanguIneos de progesterona, estradiol e hormSnio luteinizante (LH) antes e durante o estro no bovino. (Peripheral plasma levels of progesterone, estradiol and lutenizing hormone prior to and during estrus in dairy cattle.) Arq. Esc. Vet. Univ. Fed. Minas Gerais, 26: 331--337.

Chung, K.S., 1979. Effect of prostaglandin F2-alpha and nor-adrenalin on the estrus cycle and steroid hormone concentration of blood plasma in dairy cattle. Korean J. Anim. Sci., 21 : 415--433. (In Korean with English summary, tables and diagrams.)

Cooper, M.J., 1974. Control of oestrous cycles of heifers with a synthetic prostaglandin analogue. Vet. Rec., 95: 200--203.

Cooper, M.J. and Rowson, L.E.A., 1975. Control of the oestrous cycle in Friesian heifers with I.C.I. 80 ,996 , a synthetic prostaglandin analogue, structurally related to PGF2~. Ann. Biol. Anita. Biochim. Biophys., 15: 427--436.

Dean, P.D.G., Exley, D. and Johnson, M.W., 1971. Preparation of 17~-oestradiol-6-(0- carboxymethyl)oxime-bovine serum albumin conjugate. Steroids, 18: 593--603.

Dobson, H. and Dean, P.D.G., 1974. Radioimmunoassay of oestrone, oestradiol-17~ and -17~ in bovine plasma during the oestrous cycle and last stages of pregnancy. J. Endo- crinol., 61: 479--486.

Dobson, H., Cooper, M.J. and Furr, B.J.A., 1975. Synchronization of oestrus with I.C.I. 79,939, an analogue of P G F ~ , and associated changes in plasma progesterone, oestra- diol-17~ and LH in heifers. J. Reprod. Fertil., 42: 141--144.

Echternkamp, S.E. and Hansel, W., 1973. Concurrent changes in bovine plasma hormone levels prior to and during the first postpartum estrous cycle. J. Anita. Sci., 37: 1362-- 1370.

Exley, D., Johnson, M.W., and Dean, P.D.G., 1971. Antisera highly specific for 17~- oestradiol. Steroids, 18: 605---620.

Fogwell, R.L., Weems, C.W., Lewis, G.S., Butcher, R.L. and Inskeep, E.K., 1978. Secre- tion of steroids after induced luteal regression in beef heifers: effects of PGF2~ and removal of corpora lutea. J. Anita. Sei., 46: 1718--1723.

Furr, B.J.A., 1973. Radioimmunoassay of progesterone in peripheral plasma of the domest fowl in various physiological states and in follicular venous plasma. Acta Endocrinol., 72: 89--100.

106

Glencross, R.G., Munro, I.B., Senior, B.E. and Pope, G.S., 1973. Concentrations of oestra- diol-17~, oestrone and progesterone in jugular venous plasma of cows during the oestrous cycle and in early pregnancy. Acta Endocrinol., 73: 374--384.

Glencross, R.G., Esslemont, R.J., Bryant, M.J. and Pope, G.S., 1981. Relationships between the incidence of pre-ovulatory behaviour and the concentrations of oestradiol-17~ and progesterone in bovine plasma. Appl. Anita. Ethol., 7: 141--148.

Henricks, D.M., Long, J.T., Hill, J.R. and Dickey, J.F., 1974. The effect of prostaglandin F2~ during various stages of the oestrous cycle of beef heifers. J. Reprod. Fertil. , 41 : 113--120.

Karsch, F.J., Legan, S.J., Ryan, K.D. and Foster, D.L., 1978. The feedback effects of ovarian steroids on gonadotrophin secretion. In: D.B. Crighton, G.R. Foxcroft , N.B. Haynes and G.E. Lamming (Editors), Control of Ovulation. Butterworths, London, pp. 29--48.

Leaver, J.D., Glencross, R.G. and Pope, G.S., 1975. Fert i l i ty of Friesian heifers after luteolysis with a prostaglandin analogue (ICI 80996). Vet. Rec., 96: 383--384.

Louis, T.M., Hafs, H.D. and Morrow, D.A., 1974. Intrauterine administration of prosta- glandin F2~ in cows: progesterone, estrogen, LH, estrus and ovulation. J. Anim. Sci., 38: 347--353.

Peterson, A.J., Fairclough, R.J., Payne, E. and Smith, J.F., 1975. Hormonal changes around bovine luteolysis. Prostaglandins, 10: 675--684.

Rodbard, D., 1974. Statistical quality control and routine data processing for radioim- munoassays and immunoradiometric assays. Clin. Chem., 20 : 1255--1270.

Rowson, L.E.A., Tervit, R. and Brand, A., 1972. The use of prostaglandins for synchroniza- tion of oestrus in cattle. J. Reprod. Fertil. , 2 9 : 1 4 5 (Abstract).

Scaramuzzi, R.J., Turnbull, K.E. and Nancarrow, C.D., 1980. Growth of Graafian follicles in cows following luteolysis induced by the prostaglandin F2a analogue, cloprostenol. Aust. J. Biol. Sci., 33: 63--69.

Seren, E., Bono, G. and Bolelli, G., 1976. Peripheral plasma levels of oestrone, oestradiol 17~, oestradiol 17~ and oestriol in the bovine during oestrous cycle and pregnancy. Arch. Vet. Ital., 27: 155--166.

Setchell, K.D.R. and Shackleton, C.H.L., 1973. The group separation of plasma and urinary steroids by column chromatography on Sephadex LH-20. Clin. Chim. Acta, 47 : 381--388.

Smith, J.F., Fairclough, R.J., Payne, E. and Peterson, A.J., 1975. Plasma hormone levels in the cow. I. Changes in progesterone and oestrogen during the normal oestrous cycle. N.Z.J . Agric. Res., 18: 123--129.

Smith, J.F., Peterson, A.J. and Fairclough, R.J., 1979. Plasma hormone levels in the cow. II. Changes in progesterone and oestrogen after the intra-uterine administration of prostaglandin F2~ or benzyl alcohol. N.Z.J . Agric. Res., 22: 227--231.

Stellflug, J.N., Louis, T.M., Gorewit, R.C., Oxender, W.D. and Hafs, H.D., 1977. Luteolysis induced by prostaglandin F2~ before and after hysterectomy in heifers. Biol. Reprod., 17 : 535--540.

Tervit, H.R., Rowson, L.E.A. and Brand, A., 1973. Synchronization of oestrus in cattle using a prostaglandin F2~ analogue (ICI 79939). J. Reprod. Fertil. , 34: 179--181.

Thatcher, W.W. and Chenault, J.R., 1976. Reproductive physiological responses of cattle to exogenous prostaglandin F2a. J. Dairy Sci., 59: 1366--1375.

Thorneycroft , I.H., Tillson, S.A., Abraham, G.E., Scaramuzzi, R.J. and Caldwell, B.V., 1970. Preparation and purification of antibodies to steroids. In: F.G. P~ron and B.V. Caldwell (Editors), Immunologic Methods in Steroid Determination. Appleton-Century- Croft, Meredith Corporation, New York, NY, pp. 63--86.