A specific enzymeimmunoassay for progesterone in human plasma.

An enzymeimmunoassay for plasma progesterone was established using progesterone covalently linked to the enzyme, horseradish peroxidase, as the 'label'. Separation of free and bound steroid was effected by Sepharose-coupled antiprogesterone-11alpha-hemisuccinyl bovine serum albumin antiserum (Sepharose-antisera). The enzymeimmunoassay satisfied the normal criteria of specificity, precision and accuracy. Comparison of assay results obtained by radioimmunoassay (with and without thin-layer chromatography) and enzyme-immunoassay (with and without thin-layer chromatography) showed excellent agreement of results in all cases (r greater than 0.98). This enzymeimmunoassay is particularly applicable to the routine determination of plasma progesterone in the smaller clinical laboratory.     

Direct enzymeimmunoassay of progesterone in bovine milk

A sensitive enzymeimmunoassay has been developed for measuring progesterone in unextracted bovine milk. An N-hydroxysuccinimide ester of 11 alpha -hydroxyprogesterone 11-hemisuccinate has been synthesised and used to form conjugates with beta-galactosidase in buffer at pH 7.0. The degree of incorporation of progesterone into the enzyme was demonstrated using (14C)-labelled steroid and by radioimmunoassay binding inhibition. Standard curves of comparable range and sensitivity to radioimmunoassay were obtained in the presence of whole milk taken from a cow at oestrus. These advances have allowed the development of a simple micro-titre plate enzymeimmunoassay of progesterone in whole milk and will be of particular value in determination of pregnancy, prediction of the day of oestrus and diagnosis of reproductive disorders.     

Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early-pregnant ewes

Myometrial activity and plasma progesterone (P) and oxytocin (OT) were measured in early pregnant (n = 5) and cycling (n = 5) ewes. Electromyography (EMG) leads and jugular and inferior vena cava (IVC) catheters were surgically placed in ewes about 1 wk before data collection. When ewes returned to estrus, they were bred to either an intact or vasectomized ram. Continuous EMG data were collected, and blood samples were collected twice daily from day of estrus (Day 0) until Day 18. Ewes bred with an intact ram were checked surgically for pregnancy on Day 20. Computerized, quantitative analysis of EMG events showed no difference in signal from the right to left uterine horns, and no differences between pregnant and cycling ewes (p less than 0.05) until Days 14-18 when nonpregnant ewes returned to estrus and had increased EMG activity. The mean number of EMG events 180-900 s in length decreased in pregnant ewes, but this difference was not significant (p less than 0.05). Jugular plasma progesterone (P) levels confirmed corpus luteum (CL) formation in all ewes, and no differences in P between pregnant and nonpregnant ewes were measured until Days 14-18, when cycling ewes underwent luteolysis and pregnant ewes maintained CL. IVC plasma oxytocin concentrations were increased in pregnant ewes compared to concentrations in nonpregnant ewes on Days 5-13 (p less than 0.05), and the difference was largest at Day 6 (means +/- SEM pg/ml: pregnant = 68.7 +/- 13.9, nonpregnant = 30.9 +/- 19.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oestrus control and early pregnancy diagnosis in the swamp buffalo: comparison of enzymeimmunoassay and radioummunoassay for plasma progesterone

A preliminary study has been undertaken, in order to investigate the suitability of a progesterone assay in blood plasma for oestrus control and pregnancy diagnosis in the swamp buffalo (Bubalus bubalis ). Progesterone was determined both by radioimmunoassay (RIA) and by enzymeimmunoassay (EIA). Values obtained by EIA were considerably lower than values obtained by RIA. This may be partly due to the fact that only RIA values were corrected for procedural losses. Blood samples were taken on day 1 (= day of insemination) and on days 24, 27 and 30 after insemination (p.i.). Additional samples from pregnant animals were taken around day 170 p.i.. Normal progesterone values during oestrus were lower than 0.5 ng/ml, and generally the same low values were found in case of non-pregnancy at days 24, 27 and 30 p.i.. Pregnant animals showed in all cases progesterone concentrations higher than 0.5 ng/ml at days 24, 27 and 30, as well as around day 170 p.i.. These preliminary results indicate that the analysis of progesterone in plasma may be suitable for fertility control in the swamp buffalo. Furthermore we suggest that a modified EIA method can be used as a simple and rapid oestrus detection test under field conditions.     

Seasonal fluctuations in plasma progesterone concentrations in Gentile-di-Puglia and Ile-de-France ewes in southern Italy

Ile-de-France ewes had high plasma progesterone concentrations during early summer-late winter. Gentile-di-Puglia ewes had high progesterone values during the winter-spring-summer period but during autumn progesterone values were very low and oestrous behaviour was not displayed. The comparison with Ile-de-France ewes indicates that a phase shift occurs in the annual ovarian activity in ewes of the Gentile-di-Puglia breed.     

Relationship between plasma progesterone in late gestation and the incidence of dystocia in romney ewes

Thirty-eight percent (6 16 ) of the ewes with a previous history of dystocia and 15% (2 13 ) of the ewes with no dystocia in 4 lambings, had difficult births in 1976. Despite large variations in plasma progesterone concentrations among ewes at the same stage of gestation there were no differences in the progesterone concentration during the last 30 days of gestation for both single- and twin-bearing ewes with or without a history of dystocia, nor for single-bearing ewes with or without a dystocia. The progesterone concentrations declined during the last 10 days before parturition in ewes with and without dystocia. The lambs from single-bearing ewes with a dystocia did not have heavier birth weights than the lambs from single-bearing ewes without a dystocia.     

The effect of progesterone implants on ovulation and plasma levels of LH, FSH and progesterone in anoestrous ewes.

The effects of 100-mg progesterone implants in anoestrous ewes on plasma progesterone and gonadotrophin levels are described. Implant removal resulted in a surge of plasma gonadotrophins and ovulation, but there was no evidence of behavioural oestrus in 90% of the ewes. These results are discussed.     

Effect of intra-ovarian infusion of oxytocin on plasma progesterone concentrations in pregnant ewes.

The function of oxytocin receptors in the corpus luteum of pregnant ewes was investigated by infusing saline or oxytocin (100 ng/min) into the utero-ovarian artery of pregnant ewes (62 +/- 5 days, n = 12). During a 4-h infusion, plasma oxytocin (OT) concentration increased to 268 +/- 80 pg OT/ml in the OT-infused group and remained unchanged at 2.5 +/- 1.5 pg OT/ml in the saline-infused group. Progesterone concentration in jugular venous plasma (17 +/- 9 ng/ml) rapidly decreased during oxytocin infusion to 59 +/- 10% and 26 +/- 9% of control at 1.5 and 2 h, respectively; the utero-ovarian venous concentration of 64 +/- 38 ng/ml decreased by a similar magnitude during oxytocin infusion. Electron microscopy of corpora lutea, removed at the end of the experiments, showed no indication of luteolytic changes following oxytocin infusion. It was concluded that oxytocin markedly and rapidly reduces progesterone secretion in pregnant ewes.     

A simple enzymeimmunoassay of milk progesterone for monitoring fertility in dairy buffaloes

A simple, sensitive, direct (without extraction) enzymeimmunoassay (EIA) was usec to determine progesterone levels in whole milk samples of 400 Nili-Ravi dairy buffaloes. The mean progesterone values 22 d after A.I. were significantly higher in pregnant (16.6 +/- 9.2 ng/ml) than nonpregnant (below 5 ng/ml) animals. The mean progesterone values were below 0.34 +/- 0.12 (the detection limit) both at estrus and in cases of clinically diagnosed inactive ovaries, 3.18 +/- 1.9 at proestrus, 2.25 +/- 1.2 postestrus and 13.22 +/- 6.74 at Day 10 of the estrous cycle. Twenty buffaloes confirmed pregnant for 2 to 3 mo, had a mean value of 20.3 +/- 4.5 ng/ml. The EIA test is very reliable in the selection of nonpregnant buffaloes (100 %) and the confirmation of inactive ovaries and of estrus. Differential diagnosis of inactive or active ovaries can be made by analyzing two milk samples at a 7-d interval.     

Effects of heat stress on serum progesterone in cyclic ewes and on progesterone and cortisol response to ACTH in ovariectomized ewes

The daily mean of serum progesterone in cyclic ewes (N = 5) as well as the profile characteristics of progesterone and cortisol in response to an acute single dose (5 i.u./kg liveweight 0.75) of adrenocorticotrophic hormone (ACTH) into ovariectomized ewes (N = 4) was investigated during exposure to a constant thermoneutral temperature of 18 +/- 1 degree C or to a daily cyclic heat stress temperature of 18 degrees C-35 degrees C-18 degrees C, in an environmental chamber. Serum collected daily from the cyclic ewes was assayed for progesterone, while serum collected more frequently for 10 h, on the 14th day of exposure to the respective temperature, from the ovariectomized ewes was assayed for progesterone and cortisol by RIAs. In cyclic ewes, heat stress increased the area under the daily progesterone curve (P less than 0.09) but had no effect on progesterone concentration after the regression of the CL. In ovariectomized ewes, ACTH significantly elevated the response of both cortisol and progesterone (r = 0.75, P less than 0.001) within 10-15 min of injection. In the ovariectomized ewes and during heat stress, the responses of progesterone and cortisol to ACTH were characterized by an initial acute rise, a transient drop, a steep elevation and a gradual but prolonged decline. During thermoneutral temperatures, this biphasic response pattern was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique

A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.     

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.     

Plasma hormone levels and reproductive behaviour in anoestrous ewes after treatment with progesterone and PMSG

Plasma progesterone and gonadotrophin levels were studied in anoestrous ewes treated during June or July with a subcutaneous progesterone implant and/or an injection of oestradiol or PMSG. Of 32 ewes treated with progesterone during July, 9 showed a gonadotrophin surge after removal of the implant, and 10 ewes showed oestrous behaviour during the following 4 days. Six ewes conceived at this induced oestrous. Progesterone treatment during June was much less effective, with only 2 of 19 treated ewes showing a gonadotrophin surg and oestrous behaviour. Administration of PMSG at the time of implant removal in the June experiment was followed by a gonadotrophin surge and oestrous behaviour in 18 of 19 ewes, and 15 ewes conceived at the induced oestrus. An injection of PMSG, without progesterone pretreatment, stimulated a gonadotrophin surge and ovulation, but did not result in oestrous behaviour. The treatments employed appeared to initiate cyclic ovarian activity in the July experiment, but not in the June experiment.     

Radioimmunoassay of plasma oestriol

A relatively specific antiserum to oestriol-16-17-dihemisuccinate-bovine serum albumin was raised in white New Zealand rabbits and a radioimmunological method developed for the quantitative determination of ether-extractable oestriol in the peripheral venous plasma (0.1 ml) of pregnant women. The method has been evaluated and applied to the determination of plasma oestriol in 400 blood samples collected from women with uncomplicated pregnancies.     

Radioimmunoassay of plasma androsterone

A method is described and evaluated for the determination of androsterone in peripheral venous plasma (1.0 ml) from men and women. The procedure involves addition of labelled internal standard and extraction with diethyl ether. Aliquots (10 %) are removed for radioimmunoassay. An antiserum to androsterone-17-carboxymethyl oxime-bovine serum albumin and tritiated androsterone complete the system. The practical systematic errors have been determined by replicate analyses. The range of values (mean ± S.D.) in plasma from 40 healthy men are 54 ± 32 ng/100 ml, and the corresponding values for women 46 ± 28.