Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Gonadal steroid hormone level in blood plasma and milk of primiparous and multiparous nonpregnant and pregnant buffaloes

Changes in the concentration of progesterone and estradiol-17beta were measured by radioimmunoassay in 11 primiparous and 17 multiparous buffaloes at estrus and daily post insemination and in 6 nonbred buffaloes at 6 hour intervals from 4 days before expected estrus to one day after estrus. Plasma progesterone concentration at estrus was 0.1 ng/ml which rose to a peak level of 3.47 ng/ml on day 17. It fluctuated around this level in those animals which conceived, but followed a declining trend in those which failed to do so and attained lowest values on the day of next estrus. Temporal changes of the hormone revealed that the occurrence of major decline varjed between 16 and 62 h before estrus. The average concentration in milk was about three to four times higher than in plasma. The concentration of estradiol-17beta about 23.50 pg/ml at estrus and fluctuated around 10 pg/ml in animals that returned to estrus with a peak around estrus. Temporal changes of hormone revealed that peak level occurred 8-17 h before estrus. The concentration of estradiol in pregnant animals fluctuated around 10 pg/ml. The concentration in milk was about 2-3 times higher than in plasma. There was no significant (P > 0.05) difference in the concentrations of progesterone and estradiol-17beta between primiparous and multiparous animals.     

Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17β

A sensitive test system has been developed for estimation ofestradiol-17β (E₂) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E₂ is estimated by displacement of biocytinyl-E₂, that was produced by ligation of estradiol-17β, d-glucuronic acid and biocytin. Bound biocytinyl-E₂ is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to ≈120fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E₂ were found in bovine plasma: male or female calves <2.7pg/ml, cycling cow 0.5–7 pg/ml, cow during last month of pregnancy 9–310 pg/ml, mature bull 5–30 pg/ml. However, up to 1110 pg E₂/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sentitive estimation ofestradiol-17β in plasma from all type of cattle and for control of improper use of E₂ after commitment of a threshhold level.     

Relationship of embryo sexing to other methods of prenatal sex determination in farm animals: A review

Cloprostenol (ICI 80, 996) was administered to a cow with hydrallantois and a cow with hydramnios to induce parturition. In both cases circulating plasma progesterone levels at parturition were decreased from pretreatment levels. Circulating estradiol-17β levels in the case of hydramnios did not change post treatment. A 64 kg dead fetus with pituitary and adrenal defects was delivered via cesarean section. Circulating estradiol-17β levels in the case of hydrallantois rose from 200 pg/ml pre-treatment to 450 pg/ml at parturition. A live 37 kg heifer calf was delivered via forced extraction. In both cases cloprostenol was effective in reducing plasma progesterone levels, dilating the cervix and initiating labor.     

Variables associated with peripartum traits in dairy cows. V. Hormonal profiles associated with retained fetal membranes

Profiles of certain hormones measured by radioimmunoassay in 41 Holstein cows and heifers with retained fetal membranes (RFM; >12 hr postpartum) were compared to 41 peers without RFM (NRFM). Peers were matched by parity, season of calving, gestation length, dystocia and parturient paresis within prepartum diet group. Linear covariates of natural photoperiod, mean daily temperature, calf birth weight, length of gestation, and age and body weight of cow were included in the leastsquares analyses of data. Plasma profiles of prolactin and estrone were nearly identical from day 8 prepartum to day 2.5 postpartum. Plasma estradiol-17α was approximately one-third higher each day in group RFM (P<.05 across days) but estradiol-17β (Eβ) tended to be lower until day 2 prepartum (not significant). Also, plasma progesterone (P) was higher in group RFM between days 8 to 3 prepartum (p<.05 across days).Relationships between plasma P and Eβ were indicative of subsequent RFM (>24 hr rather than >12 hr), but only on day 6 prepartum. Three of four cases of 12 to 24 hr RFM had P and Eß profiles similar to NRFM. Either a combination of low P (<3.0 ng/ml) and low Eß (<100 pg/ml) or only high P (>7.9 ng/ml) were associated with a ten-fold higher rate of RFM (>24 hr) than when P was intermediate (4 to 8 ng/ml) and Eß exceeded 99 pg/ml. These results are in agreement with prior data wherein RFM were induced at premature births either by ovariectomy during pregnancy or by glucocorticoids.     

Seasonal variation in serum levels of steroid hormones and their relation with seminal quality and libido in buffalo bulls

Blood samples from 15 breeding male Murrah buffaloes were collected during the winter, summer and monsoon seasons. Seminal characteristics and sexual behaviour were also studied. Serum samples were analysed for testosterone, progesterone and estradiol-17beta levels by radioimmunoassay. The studies showed significantly lower values for testosterone during winter (0.53 +/- 0.06 ng/ml) than during summer (1.22 +/- 0.19 ng/ml) and monsoon (1.06 +/- 0.12 ng/ml). The progesterone level was lowest during monsoon (84 +/- 9 pg/ml), intermediate during winter (115 +/- 14 pg/ml) and highest during summer (224 +/- 24 pg/ml). The mean level of estradiol-17beta was almost double (9 +/- 0.7 pg/ml) during monsoon as compared to winter (5 +/- 0.1 pg/ml). The correlations between hormone levels, seminal characteristics and sexual behaviour were of low magnitude.     

Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor.

Oxytocin receptors were measured in myometrium and intercaruncular endometrium of cows during pregnancy and parturition. Concentrations of estradiol-17 beta, estrone, and progesterone in peripheral blood were also measured. Receptor concentrations in the endometrium rose almost 200-fold from Day 20 to term (p < 0.0001, ANOVA), from 40 +/- 11 to 7300 +/- 1430 fmol/mg protein. Myometrial receptor concentrations increased 10-fold from 180 +/- 36 fmol/mg on Day 20 to 1850 +/- 360 fmol/mg protein at term (p < 0.0001, ANOVA). During labor, endometrial receptors (6600 +/- 1300 fmol/mg) remained at prelabor values, whereas myometrial receptor concentrations had decreased to 1190 +/- 316 fmol/mg (not significant) and declined further postpartum. Plasma concentrations of progesterone declined from 4-5 ng/ml to about 2 ng/ml between Days 250 and 282 and dropped to < 0.2 ng/ml shortly before delivery. Plasma concentrations of estrone and estradiol-17 beta were below 10-20 pg/ml until Day 230. Estrone concentrations were significantly (p < 0.05) increased by Day 250 and estradiol-17 beta by Day 270, and then both rose rapidly. During labor, plasma estrone was 1135 +/- 245 pg/ml and plasma estradiol-17 beta was 226 +/- 131 pg/ml. The molar ratio of estrone and estradiol-17 beta to progesterone rose from less than 0.01 to 4.4 during labor, and was correlated with oxytocin receptor concentrations in endometrium (r = 0.5160, p < 0.001), but not those in myometrium (r = 0.0122). The regulation of oxytocin receptors by ovarian hormones in the two tissues may therefore differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of the vitelline envelope precedes the active uptake of vitellogenin during oocyte development in the rainbow trout,Oncorhynchus mykiss

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. lmmunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 μm. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17β increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 μm) present just inside the oolemma, when the oocytes reached a diameter of 600 μm. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol 17β levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 μm had an immunoreactive vitelline envelope with a thickness of about 3 μm. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17β observed in females are of physiological significance. © 1994 Wiley-Liss, Inc.     

Comparable testosterone responses are produced by constant infusion of LH or GnRH in normal men

Luteinizing hormone (LH) was infused continuously at a rate of 1.3 IU/min to 4 normal adult men. A 4 to 5-fold increase in serum LH was noted by 8 hours. Serum FSH declined steadily throughout the infusion period in the face of rising concentrations of gonadal steroids. Basal plasma testosterone of 4.7 +/- 0.4 ng/ml rose progressively to a peak of 11.1 +/- 0.9 ng/ml at hour 56 (p less than 0.005). A similar pattern was demonstrated by plasma androstenedione. Plasma 17 alpha-hydroxyprogesterone rose from a basal concentration of 0.81 +/- 0.14 ng/ml to a peak concentration of 2.6 +/- 0.3 ng/ml at hour 36 of the infusion and subsequently declined. A similar course was followed by serum estradiol-17 beta, which achieved a maximal concentration of 70.0 +/- 10.4 pg/ml at hour 36. Results are compared to those obtained with continuous infusion of GnRH in normal adult men. Testosterone responses were similar, whereas elevations in 17 alpha-hydroxyprogesterone and estradiol were higher following GnRH infusion. This difference may be consequent upon a direct gonadal effect of GnRH, or may be secondary to local regulation of testicular steroidogenesis by estradiol-17 beta.     

Luteinizing hormone concentrations in anestrous ewes administered various estrogens

Twenty four anestrous ewes were evenly assigned to one of six groups and administered either sesame oil, estradiol-17β, estradiol-17α, estrone, estradiol benzoate or estradiol valerate. All estrogen treated ewes received 50 μg of the respective estrogen. Blood plasma was collected for 28 hours post-treatment and quantified for luteinizing hormone (LH) by radioimmunoassay. An estrogen induced LH surge was detected in at least three of the four ewes administered either estradiol-17β, estrone, estradiol benzoate or estradiol valerate whereas only one of the four estradiol-17α treated ewes and none of the ewes administered sesame oil had an LH surge. The interval from treatment to peak LH was similar for estradiol-17β (17.3±2.7 hours), estrone (18.5±1.0 hours) and estradiol benzoate (19.0±0.6 hours) treated ewes but delayed 7 to 9 hours for ewes administered estradiol valerate (26.0±1.2 hours).     

Male pseudohermaphroditism due to testicular 17-ketosteroid reductase deficiency.

A new case of testicular 17 ketosteroid reductase (17 KSR) deficiency without gynecomastia was investigated. Delta4 androstenedione (15.6 ng/ml) was ten times the normal range, unchanged after dexamethasone administration. In contrast, plasma testosterone (4.1 ng/ml) was in the low normal male range and plasma dehydroepiandrosterone (4.2 ng/ml) was normal. Plasma luteinizing hormone and follicle-stimulating hormone were increased (162 and 470 ng/ml LER 907 respectively). After adrenal suppression and human chorionic gonadotropin stimulation, the increase of delta4 androstenedione was in contrast with the inertia of testosterone. In spermatic venous plasma delta4 androstenedione level (293.2 ng/ml) was very high and testosterone level (7.1 ng/ml) a hundred times below the normal mean. Plasma estrone (124 pg/ml) was increased and estradiol (22 pg/ml) was normal. In spermatic venous plasma estrone was elevated and estradiol very low (1380 and 32 pg/ml respectively). It is the third case of 17 KSR deficiency where the lack of E2 increase explains the absence of gynecomastia.     

Direct radioimmunoassay of estradiol-17β in defatted bovine milk

A radioimmunoassay was developed for rapid determination of estradiol-17β concentrations in unextracted defatted bovine milk. The assay was dependent on the use of a highly specific anti-estradiol-17β antiserum. Application of a formula to correct for the interference associated with individual milk samples and use of appropriate assay blanks facilitated interpolation on a buffer standard curve. The assay offered a high degree of sensitivity (0.6pg/ml milk) and a precision (within-assay coefficient of variation: 0.196; between-assay CV:0.191) comparable with contemporary extraction methods.     

Effect of estradiol and relaxin on growth of fibroblastic cells isolated from guinea pig mammary glands

Fibroblasts were isolated from the mammary glands of guinea pigs and grown in 96-well culture plates. They were treated with a factorial arrangement of porcine relaxin (0.0, 0.5, 1.0 or 1.5 micrograms/ml) and estradiol-17 beta (0, 200, 400 or 600 pg/ml). Tritiated thymidine or uridine was added to a final activity of 25 nCi per well and the cells incubated at 37 degrees C for 48 h. Cells were then harvested onto filter paper and counted for tritium. Controls (0.0 micrograms/ml relaxin and 0 pg/ml estradiol) incorporated 3.7 nCi of tritiated thymidine and 4.8 nCi tritiated uridine. Both relaxin and estradiol altered the incorporation of thymidine and uridine. There was also an interaction between the two hormones. Thymidine incorporation with no estradiol and 1.5 micrograms/ml relaxin was 129% of controls. The optimum incorporation of thymidine occurred with 0.5 micrograms/ml relaxin and 400 pg/ml estradiol. This combination of hormones gave a response of 145% of controls. Uridine incorporation followed a different pattern. Relaxin alone at a concentration of 1.5 micrograms/ml gave a near-optimum response of 141% of control. The optimum combination of relaxin and estradiol for uridine incorporation was 1.5 micrograms/ml relaxin and 400 pg/ml estradiol, which gave a response of 156% of controls. These data indicated that relaxin and estradiol alter DNA and RNA synthesis in mammary fibroblasts and thus may be important in controlling the growth of the mammary gland stroma.     

Leptin concentration in plasma and in milk during the interpartum period in the mare

The aim of this work is to investigate on plasma profiles of leptin and estradiol 17beta during the interpartum period and leptin concentrations in the milk and in the colostrum during the period from parturition to the successive delivery in mare. Leptin plasma concentration varied from 5.1+/-2.3 ng/ml after the first parturition (week 0) to 3.0+/-0.7 at week 21 (p<0.05), then it increased to maximal level at week 49 (6.9+/-1.0 ng/ml, p<0.05). Leptin concentration in the colostrum and in the milk has been significantly (p<0.05) higher than that in plasma samples at week 1 (milk 8.8+/-2.3 versus plasma 5.2+/-0.6 ng/ml) and between week 12 and 17. This difference may be explained with a local leptin production at mammary level and supports a role of leptin in the mammary gland and/or in foal intestine. Estradiol 17beta increased from week 15 (17.9+/-2.3 pg/ml) up to 487.9+/-67.7 pg/ml at week 43. Plasma estradiol 17beta rise anticipated by 4 weeks plasma leptin increase and it does not seem to be positively correlated to leptin secretion.     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.     

Non-invasive monitoring of ovarian function in several felid species by measurement of fecal estradiol-17β and progestins

An extraction and assay procedure to measure fecal estradiol-17β and progestin concentrations in several cat species was developed and validated for use for noninvasive monitoring of ovarian function. Fecal samples were collected over a range of 3–20 months from female tigers (three), lions (three), snow leopards (three), cheetahs (two), caracals (two), and domestic cats (five). Samples were extracted with 90% methanol, lipids removed with petroleum ether, and the estradiol and progestins in the methanol measured by radioimmunoassay (RIA). High Performance Liquid Chromatography (HPLC) fractionation and subsequent RIA of the fractions indicated that the estradiol-17β antiserum cross-reacted primarily with estradiol-17β in the feces of lions and tigers and was assumed to be specific for estradiol-17β in the feces of other species as well. However, there were several immunoreactive compounds, presumably progesterone metabolites, excreted in the feces which varied both quantitatively and qualitatively among species. The behavior of tigers, lions, cheetahs, and caracals was visually monitored during the collection period and frequency of sexual behaviors was positively correlated with increases in fecal estradiol in all species observed. The mean fecal estradiol-17β peaks were as follows: tigers, 128.0 ± 13.1; lions, 186.0 ± 14.8; snow leopards, 136.7 ± 15.9; cheetahs, 140.9 ± 9.0; caracals, 24.5 ± 4.0; and domestic cats 158.9 ± 19.3 ng/gm. Fecal progestin concentrations rose significantly (P < 0.001) only after breeding or during pregnancy and were as follows: tigers, 5.6 ± 0.6; lions, 1.9 ± 0.1; cheetahs, 8.4 ± 1.1; and caracals, 2.4 ± 0.4 μg/gm. Fecal progestins were elevated for one-half to two-thirds of the gestation length during presumed pseudopregnancy but remained elevated throughout successful pregnancies. These results suggest that ovarian function can be monitored noninvasively in the family Felidae by the measurement of fecal estradiol-17β and progestin concentrations. © 1995 Wiley-Liss, Inc.     

Steroidogenic capabilities of the early mouse embryo

Preimplantation embryos from ICR albino mice were used to determine progesterone and estradiol-17β production during incubation in BMOC-2. Following culture of 40 embryos/culture at either the morula, early blastocyst or late blastocyst stages, progesterone and estradiol-17β contents were 192±27 and 82±22 pg, 289±50 and 147±46 pg and 157±28 and 88±23 pg, respectively, for incubated samples and 306±68 and 89±40 pg, 404±63 and 125±44 pg, and 241±54 and 86±39 pg, respectively for control samples. Although, there were significant stage of development and treatments effects (P<0.05) for progesterone, production of this steroid was not evident. These data suggest that the early preimplantation mouse embryo does not produce progesterone or estradiol-17β in a defined culture system.     

Sex hormones correlated with sex skin swelling and rectal temperature during the menstrual cycle of the pigtail macaque (Macaca nemestrina).

Daily measurement of serum luteinizing hormone, estradiol-17beta, and progesterone were made during the menstrual cycle in nine pigtail macaques (Macaca nemestrina). All data were normalized to the day of the luteinizing hormone peak. Serum estradiol-17beta increased from approximately 100 pg/ml during the early follicular phase to 442 +/- 156 pg/ml during the maximum midcycle concomitant with the luteinizing hormone peak, and a small increase in serum estradiol-17beta was observed during the luteal phase coincident with the progesterone peak. Serum progesterone values increased slightly at the time of the luteinizing hormone peak and increased from 0.2-0.3 ng/ml during the midfollicular phase to peak levels of 8.3 +/- 1.75 ng/ml 9 days after the luteinizing hormone surge. Serum luteinizing hormone remained low and relatively constant throughout the early and midcycle, then sharply increased approximately four-fold to peak values of 6.25 +/- 0.9 ng/ml. Sex skin swelling increased slowly during the follicular phase and declined slowly throughout the early luteal phase. Rectal temperature did not change significantly throughout the menstrual cycle. The similarity of plasma sex hormone changes during the menstrual cycle between women and the pigtail macaque suggested that this nonhuman primate should be a useful animal model for studying human reproduction.     

Plasma estradiol 17-sulfate and 2-hydroxyestradiol 17-sulfate levels and their metabolic clearance rates in rats

By using highly specific antisera against estradiol 17-sulfate (E2-17-S) and against 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S), plasma concentrations of these sulfates in Wistar rats were determined. The plasma levels of E2-17-S and 2-OH-E2-17-S in the male were 23.5 +/- 5.3 and 21.6 +/- 6.2 pg/ml, respectively. During the estrus cycle of the female, the plasma concentration of E2-17-S reached its highest level 69.0 +/- 11.8 pg/ml, during the diestrus stage, and its lowest level 36.9 +/- 6.6 pg/ml, during the proestrus stage. Similar tendencies were observed in the case of 2-OH-E2-17-S. To examine the dynamic behavior of both sulfates, the plasma metabolic clearance rate (MCRp) of E2-17-S and 2-OH-E2-17-S were determined by infusion experiments. MCRp of E2-17-S and 2-OH-E2-17-S in male rats were 102 and 653 ml/h (means), respectively, and in female rats were 115 and 644 ml/h (means), respectively. The low MCRp values of both sulfates imply their slow metabolic turn-over.     

Parturition in beef cows following administration of porcine relaxin at ten days prepartum

Administration of procine relaxin (pRLX) to heifers 5 d prepartum has been reported to expedite parturition. Thirty-eight mature crossbred beef cows were randomly assigned to one of three treatment groups. Control animals (C; n = 13) received an intramuscular (i.m.) injection of 2 ml corn oil and 2 ml i.m. phosphate-buffered saline (PBS) 24 h later; relaxin treated animals (RLX; n = 13) received 2 ml i.m. corn oil and 1.0 mg i.m. pRLX 24 h later; estradiol-relaxin treated animals (E-RLX; n = 12) received 20 mg i.m. estradiol benzoate (EB) and 1.0 mg i.m. pRLX 24 h later. Treatment with pRLX occurred at 272.6+/-0.14 d of gestation. The pRLX had been purified to homogeneity from porcine ovaries collected during late pregnancy and was determined to have >/=3000 U/mg by the mouse interpubic ligament bioassay. Peripheral blood samples were collected from all cows at 0, 4, 8 and 24 h, respective to corn oil or EB administration, and assayed for plasma estradiol-17beta. At 24 h post administration of EB, plasma estradiol-17beta concentrations were 48.0+/-10.5 pg/ml for C and RLX cows and 178.5+/-14.8 pg/ml for E-RLX cows. There were no treatment effects (P>/=0.10) for elapsed time from treatment to parturition (304.2+/-22.4 h), gestation length (285.2+/-0.9 d), calving difficulty score (1.05+/-0.04), calf vigor score (1.05+/-0.04) or calf birth weight (38.0+/-0.88 kg). Additionally, there were no retained placental membranes in any cows. Administration of pRLX intramuscularly to beef cows at 10 d before expected parturition was not effective in inducing premature parturition. Furthermore, the effectiveness of pRLX in inducing parturition was not enhanced by pretreatment with estradiol benzoate.