Circulating gonadotrophins and follicular dynamics in anestrous ewes after treatment with estradiol-17beta.

Plasma FSH, LH, estradiol (E2) and progesterone (P4) profiles and patterns of follicular growth and regression by ultrasonography were determined after E2 treatment (1 microg/kg) in anestrous ewes. Fifteen ewes were treated with one (group I, n=7) or two (group II, n=4) i.m. injections of E2 with a 24h interval, or two oil injections with a 24h interval (group C, n=4). Blood samples for E2, P4, FSH and LH determinations were collected daily 4 days before the initiation of the treatment (day 0), when bleeding increased to every 2h starting 2h before treatment until 56h after the first injection and from then on every 6h until day 8, and twice per day till the end of the experiment (day 9). During the experimental period (days -4 to 9), transrectal ultrasonic examinations were carried out daily using a 7.5 MHz linear array probe. Number and size of follicles > or =3mm in diameter were recorded. No estrous was detected before, during or after treatment. LH and FSH surges were observed 10-18h after the first E2 injection. The second E2 injection stimulated another release of LH but no surges. E2 inhibited FSH levels before the surge and the second E2 injection induced a longer inhibition. No ovulation was detected by ultrasonography during the experimental period and P4 levels remained low (<0.7 nmol/l) before, during and after the treatment in all ewes. There was an effect of E2 treatment on the diameter of the largest follicle, a decrease could be observed 3 days after the first injection in both ewes of groups I and II. The E2-treated groups had a higher frequency of ewes showing wave emergence on day 3 (day 1.5+/-1,2.4+/-0.4 and 2.5+/-0.5 for control, groups I and II). LH and FSH surges were observed after E2 treatment, but were not able to provoke ovulation neither luteinization. In contrast, the treatment was associated with the regression of the largest follicle and with emergence of a new follicular wave on day 3.     

Ovarian follicular dynamics after cauterization of the dominant follicle in anestrous ewes

An experiment was conducted to ascertain if follicles could reach ovulatory size after the largest follicle (dominant) has been removed at different times during a progestin treatment in anestrous ewes, and secondly to determine if these new follicles could respond to an hCG-induced ovulation and have similar function as corpora lutea. Mature crossbred sheep (n=44) in anestrous were treated with an intravaginal sponge containing 40 mg of FGA (day 0=sponge insertion) for 9 days. Treatments consisted of cauterization of the largest follicle on the experimental day 3 (T1), day 6 (T2) and day 9 (T3); day 12 to ascertain the size of the largest follicle in control ewes. During laparotomies, the diameters of the largest follicle (DF), and those of the second and third largest follicles (SF1 and SF2, respectively) were determined. On day 12, a second laparotomy was performed for those ewes which had their DF cauterized on days 3, 6 and 9, a fourth group was left intact and only laparotomized on day 12. At this time, the size of the new DF, SF1 and SF2 were determined. Immediately after the laparotomy on day 12, all the ewes were treated with 1000 i.u. of hCG to induce ovulation. Blood samples were collected daily from day 0 to 50 and samples were analyzed for progesterone concentrations. The size of the DF at the time of sponge removal was smaller that those observed on day 3 or 6 of sponge suggesting that follicles in ewes treated with this progestin regress and a new wave of follicular development ensues between day 6 and the time of sponge removal. The size of the DF on day 12 was also smaller in ewes that have the largest follicle removed at the time of sponge removal reflecting that these follicles had a shorter period of growth; however, the rate of growth was greater for these follicles than for follicles arising after cauterization on day 3 or 6 after sponge insertion. There were no differences among treatments, in the number of ewes that formed a corpus luteum (CL) in response to hCG. Life span of the corpora lutea did not differ among ewes having their DF removed on day 6 or 9 or those that served as controls, however, ewes that had their DF removed on day 3 developed longer lived CL in a larger proportion of animals. Average progesterone concentration during the life span of the induced corpora lutea was greater in control ewes than in any other experimental group. These observations allow us to conclude that, (a) the follicular dynamics observed in anestrous ewes treated with a progestin intravaginal sponge resembles that observed during the normal estrous cycle in the ewe; (b) the effects of progesterone on life span of the corpus luteum could not be only related to direct effects at the follicle but also involve changes in other components of the uterine-ovarian-hypothalamic axis; (c) the mechanisms controlling luteal life span seem to be different to those mechanisms controlling the function of the induced corpus luteum.     

Suppression of follicle wave emergence in cyclic ewes by supraphysiologic concentrations of estradiol-17beta and induction with a physiologic dose of exogenous ovine follicle-stimulating hormone

Follicle waves are preceded by follicle-stimulating hormone (FSH) peaks in ewes. The purpose of the present study was to see whether estradiol implant treatment would block FSH peaks to create a model in which the effect of the timing and mode of FSH peaks could be studied by ovine FSH (oFSH) injection. In Experiment 1, 10 ewes received estradiol-17beta implants on Day 4 after ovulation (Day 0, day of ovulation); five ewes received large implants, and five ewes received small implants. Five control ewes received empty implants. In Experiment 2, 12 ewes received large implants on Day 4. On Day 9, six ewes received oFSH twice, 8 h apart (0.5 microg/kg; s.c.). Implants were left in place for 10 days in both experiments. In both studies, ovarian ultrasonography and blood sampling was done daily. In Experiment 1, estradiol concentrations were significantly higher in ewes with large implants (10.4 +/- 0.7 pg/ml) compared with controls (3.9 +/- 0.7 pg/ml) and ewes with small implants (5.4 +/- 0.7 pg/ml; P < 0.001). A significant reduction was found in mean FSH peak concentration (31%; P < 0.05) and FSH peak amplitude (45%; P < 0.05) in ewes with large implants compared with controls. Mean and basal FSH concentrations were unaffected by the large implants. The large implants halted follicle-wave emergence between Day 0 and 8 after implant insertion. The small follicle pool (2-3 mm in diameter) was unaffected by the large implants. When oFSH was injected into ewes with large implants, a follicle wave emerged 1.5 +/- 0.5 days after injection; however, in ewes given saline alone, a follicle wave emerged 4.8 +/- 0.8 days after injection (P < 0.01). We concluded that truncation of FSH peaks by estradiol implants prevented follicle-wave emergence, but injection of physiologic concentrations of oFSH reinitiated follicle-wave emergence.     

Response of anestrous ewes to norgestomet and PMSG

A 10-day treatment regime with a subcutaneous ear implant containing 3 mg of norgestomet, accompanied by an intramuscular injection of 1.5 mg norgestomet and 0.5 mg estradiol valerate (EV) on day 1 and 750 I. U. pregnant mares serum gonadotropin (PMSG) given intravenously on day 10, proved effective in eliciting estrus in 72% of 110 anestrous ewes within 5 days of treatment. Ewes which were treated in months closer in proximity to the normal breeding scason responded with significatly increased induction of estrus, with 71, 37, 59, 74, and 97% in estrus for ewes which were treated in February through June, respectively. Comparable estrous response in nontreated, control ewes was 0, 13, 0, 10, and 24% during February through June, respectively. (Treated vs controls, P<.01). Pregnancy rate to first service of ewes in estruc was 51% in treated and 30% in control ewes (P>.10). Overall pregnancy rate for all ewes in both groups was 36% in treated and 3% in control ewes during 5 or 16 days of breeding, respectively (P<.01).     

Markers of atresia in ovarian follicular components from rhesus monkeys treated with estradiol-17 beta

A new model for the investigation of atresia in rhesus monkeys is presented. This model is based upon the reliable induction of an atresia-like process in the dominant preovulatory follicle (DF) by estradiol-17 beta administered subcutaneously via Silastic capsules for 24 h. Data obtained from follicular contents aspirated from treated animals demonstrated alterations in the putative markers of atresia similar to those described in other models of atresia. Although follicle size and appearance and volume of follicular fluid (FF) were unaltered in treated animals, FF was much more viscous than that aspirated from follicles in untreated animals; this was apparently due to a greater quantity of intercellular matrix that was sensitive to digestion by hyaluronidase. In treated animals, FF concentrations of estrogen (E) and progesterone (P) were depressed 3- and 6.6-fold, respectively. Viability of granulosa cells (GC) from these animals was reduced by 40%, as was their ability to release basal amounts of E and P in vitro. Accumulation of P by GC from treated animals approximated unstimulated control levels when human follicle-stimulating hormone (hFSH) was included in the culture. Therefore, FSH may have a limited capability to "rescue" GC from atresia induced by estradiol. The percentage of cells that bound 125I-hFSH maximally, as measured by autoradiography following 72 h in culture, was not altered by treatment. Oocytes from animals treated with estradiol showed signs of degeneration at aspiration, and deteriorated further in culture. This model is unique in that atresia can be induced in the single DF of a primate species, and thus avoids the disadvantages inherent to studying atresia of heterogeneous follicles in polytocous species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistent infertility in ewes after prolonged exposure to oestradiol-17 beta

Merino ewes were treated with implants which released 300 micrograms oestradiol-17 beta per day or 5 mg progesterone per day, or both, for 9 months (Months 1-9), and after an 11-month intermission were treated again for 6 months (Months 20-26). Ewes were run with rams at Months 16, 28 and 40. Fertility was not affected by the first exposure period, but the second exposure to oestradiol reduced the fertility of ewes at both subsequent mating periods. Affected ewes returned to service more frequently (P less than 0.01) and were less likely to conceive (P less than 0.05). After mating, a normal population of spermatozoa was established in the caudal cervix, but transport through the cervix was impaired in affected ewes and there were fewer spermatozoa (P less than 0.01) in the cranial cervix. In affected ewes, the spinnbarkeit of cervical mucus was reduced (P less than 0.05), and the histological appearance of the cervix changed, looking like that of the uterus. Treatment with progesterone did not affect fertility, cervical mucus or sperm transport, but diminished the histological abnormalities produced by oestradiol (P less than 0.05). These results show that oestradiol-17 beta given after puberty can cause the same kind of permanent sexual transdifferentiation that is produced by the oestrogenic isoflavones in ewes with clover disease. The results suggest that this change may require more than a single exposure to oestrogen.     

Response of ovariectomized ewes to injection of oestradiol-17 beta at different times of the year

Long-term ovariectomized Clun Forest ewes were challenged with a range of doses (12.5-50.0 micrograms/injection) of oestradiol benzoate every 2 months from March to November. All treatments induced a biphasic pattern of change in LH concentrations, consisting of an initial depression in concentrations followed by an LH peak, similar to a preovulatory LH surge. The positive feedback response to 12.5 micrograms oestradiol was significantly lower than that after the two higher dose levels, but the magnitude of the response showed no significant seasonal variation. It is concluded that a seasonal change in responsiveness to positive feedback is unlikely to contribute to the absence of ovulation during seasonal anoestrus.     

Effects of lactation status, progestogen and ram exposure on response to cloprostenol in ewes during the anestrous season

Progestogen pretreatment and introduction of rams were used to prepare 432 Rasa aragonesa ewes for synchronization of estrus with prostaglandin (PG) during anestrus. The experiment was a 3 x 3 x 2 factorial with lactation status, ram/progestogen treatment and treatment with pregnant mare's serum gonadotropin (PMSG; 250 IU) at injection of PG as main effects. At ram introduction (Day 0), ewes were dry (Group 1), weaned (Group 2) or suckling a lamb (Group 3). They received either norgestomet implants for 12 d (Days -12 to Day 0 (Group A), ram introduction for 12 d (Days 0 to 12) (Group B) or both (Group C). Half the ewes received PMSG with PG (100 mug Cloprostenol) on Day 12. Pregnancy rate was higher at first service in dry (Group 1; 42%) than in recently-weaned ewes (Group 3; 24% and Group 2; 31%; P<0.01). Occurrence of estrus and conception and pregnancy rates to first service were higher (P<0.01) in ewes previously exposed to rams (Groups B and C) than in ewes treated only with progestogen (Group A). There were no effects of PMSG, no interactions among the three variables and no differences in prolificacy (1.12 at first service).     

Local versus systemic effects of exogenous estradiol-17 beta on ovarian follicular dynamics in heifers with progestogen implants

Two experiments were designed to determine if the suppressive effect of estradiol treatment on ovarian follicles in progestogen-implanted heifers is mediated directly at the ovary or systemically, at a higher level. The purpose of Experiment 1 was to determine a minimal effective dose of estradiol-17beta (E-17beta) that would induce follicle regression in progestogen-implanted heifers. Beef heifers were implanted with progestogen on Day 2 (Day 0=ovulation) and were assigned randomly to five groups: control (sesame seed oil, n=9); 0. 1 mg of E-17beta (n=8); 0.5 mg of E-17beta (n=8); 1 mg of E-17beta (n=8); or 5 mg of E-17beta (n=8) by intramuscular (im) injection on Day 3. Treatment with 5 and 1 mg of E-17beta resulted in smaller (P<0.05) day-to-day diameter profiles of the dominant follicle compared with controls, whereas 0.1 mg of E-17beta did not have an apparent effect on follicle growth. The effect of a dose of 0.5 mg was intermediate and tended (P<0.06) to result in a smaller diameter profile of the dominant follicle compared with control heifers. Experiment 2 was designed to utilize a subminimal dose of E-17beta (0.1 mg), locally, to determine whether estradiol treatment induces follicle regression through a direct action on the ovary. Beef heifers received a progestogen ear implant on Day 2 and were assigned randomly to five groups on Day 3: control (sesame seed oil, n=8); 5 mg of E-17beta im (n=8); 0.1 mg of E-17beta im (n=8); 0.1 mg of E-17beta given into the wall of the uterus, near the tip of the horn ipsilateral to the dominant follicle (intrauterine (iu), n=8); or 0.1 mg of E-17beta given into the stroma of the ovary, immediately adjacent to the dominant follicle (intraovarian (io), n=6). Local (iu and io) treatments were given via a transvaginal ultrasound-guided needle injection. Treatment with 5 mg of E-17beta im resulted in suppression of the dominant follicle of the first follicular wave and early emergence of the second follicular wave (P<0.05). Diameter profiles of the dominant follicle in heifers treated with 0.1 mg im or 0.1 mg iu differed from those of control heifers on Day 5, whereas diameter profiles of the dominant follicle in heifers treated with 0.1 mg io did not differ from the controls. Daily changes in diameter of the dominant follicle did not differ among the three groups treated with 0.1 mg of E-17beta (im, iu and io). Hourly changes in circulating concentrations of FSH and LH were not detected following estradiol treatment either before or after the results were combined for all estradiol-treated groups. Results are supportive of the hypothesis that the suppressive effect of estradiol in cattle is exerted indirectly through a systemic route rather than directly at the ovary. Although low plasma concentrations of FSH and LH were not detected, systemic treatments with high E-17beta dosages resulted in follicular suppression whereas local treatments with subminimal dosages, within the ovary bearing the dominant follicle, were without effect.     

Comparison of the methylation of estradiol-17 beta and of estradiol-17 alpha by dimethyl sulfate

E₂-17β and E₂-17α give substantially similar yields of 3-methyl ether and dimethyl ether when methylated by Brown's procedure.     

Progesterone and estradiol-17beta in the peripheral plasma of Brahman cows with single and twin pregnancies

Peripheral plasma samples from Brahman cows with single and twin pregnancies were assayed for progesterone and estradiol-17beta throughout pregnancy. The twin pregnancies were obtained by transfer of Friesian embryos to inseminated single-ovulating Brahman cows. The twin-bearing cows had significantly higher levels of progesterone at 8 and 36 weeks of pregnancy. There were no differences in estradiol-17beta levels until the pre-parturient rise which occurred earlier in twin pregnancies. Intra-muscular injection of progesterone had no measurable effect on peripheral plasma levels of estradiol-17beta in Charolais cows 31 - 32 weeks pregnant.     

Diurnal variation in the response of anoestrous ewes to the ram effect

The re-introduction of rams after a period of separation was used to stimulate LH secretion and induce ovulation in seasonally anovulatory ewes maintained under natural photoperiod. In 2 experiments, the rams were introduced in the morning or the evening to test for diurnal variations in responsiveness to the treatment. In the first experiment, with Romanov ewes, the ram-induced increase in tonic LH secretion was significantly earlier in the ewes treated (N = 6) at 07:30 h (mean +/- s.e.m. delay to first pulse: 20 +/- 6 min) than in those (N = 5) treated at 19:30 h (66 +/- 15 min; P = 0.006). The pulse interval after the ram effect was significantly shorter in ewes that subsequently ovulated (120 +/- 10 min) than in ewes that did not ovulate (288 +/- 108 min; P = 0.043). There was a significant decline in pulse amplitude from 6.7 +/- 1.2 to 3.4 +/- 0.6 ng/ml (both groups combined) after the introduction of rams (P = 0.040). Of the 11 ewes, 7 subsequently ovulated and a preovulatory LH surge was observed in 6 of these 30-36 h after ram introduction. In the second experiment, with seasonally anoestrous Préalpes-du-Sud ewes, the effect of the timing of the introduction of rams on the periovulatory events was tested. The delay to the onsets of oestrus and the LH surge was not affected, but the ovulation rate was higher after ram introduction in the morning (1.42) than in the evening (1.14). In the 12-h period before the introduction of the rams in the first experiment, there was a difference between the groups in the secretion of LH, but the existence of diurnal rhythms in the concentrations of LH or FSH were not confirmed in a later study in which 7 ewes were sampled every 20 min for 36 h. In contrast, there was a distinct diurnal variation in the secretion of prolactin, with the highest values being recorded at night and the lowest around midday (P = 0.025). The rise and fall in prolactin values did not appear to coincide with dawn or dusk.(ABSTRACT TRUNCATED AT 400 WORDS)     

The preparation of estradiol-17 beta sulfates with triethylamine-sulfur trioxide

Reaction of estradiol-17 beta with triethylamine-sulfur trioxide in pyridine gives exclusively monosulfation at the C17-hydroxyl group with the preparation of 17 beta-sulfooxyestra-1,3,5(10)-trien-3-ol triethylammonium salt (V). The structural assignment suggested by spectroscopic measurements was confirmed by synthetic studies. (Formula: see text) A synthesis of 3-sulfooxyestra-1,3,5(10)-trien-17 beta-ol triethylammonium salt (II) has been accomplished based on the preparation of 17 beta-formyloxyestra-1,3,5(10)-trien-3-ol (XIII). Fusion of the 3-sulfate triethylammonium salt II gives rise to the 17-sulfate triethylamine salt V. The preparation of estradiol-17 beta disulfate has also been achieved.     

Ovulation rate in ewes after single oral glucogenic dosage during a ram-induced follicular phase

A 2-factor factorial array with three replicates (N = 280) was used to simultaneously assess the effects on ovulation rate of two alternative doses of medroxy-progesterone acetate (MPA) (10 or 60 mg), applied during a 6-day priming period, and the effect of a single dosage of a glucogenic formulation, administered immediately before ram exposure to groups of adult seasonally anovular Corriedale ewes. The glucogenic formulation contained 1,2,3-propanetriol (glycerol; 70% $\text{v}\text{v}$), 1,2-propanediol (propylene glycol; 20% $\text{v}\text{v}$) and distilled water (10% $\text{v}\text{v}$). At sponge withdrawal, a single oral dose of 100 ml of this formulation or the same volume of distilled water was administered to treated and control groups, respectively, and ewes were immediately exposed to rams and hormonally-induced oestrous ewes. Data from an ancillary experiment (n = 10) showed significantly (P < 0.005) above normal plasma glucose levels in treated animals at 3 and 6 h after dosage. A significant interaction (P = 0.0006) between MPA priming doses and glucogenic supplementation was detected. Supplemented ewes, among those exposed to the lower dose of MPA, exhibited a higher (P = 0.0098) mean ovulation rate (1.56 ± 0.076) than ewes that did not receive glucogenic treatment (1.31 ± 0.060). In contrast, ovulation rate was significantly decreased (P = 0.021) from 1.30 ± 0.058 to 1.13 ± 0.042 after glucogenic treatment in ewes that were primed with sponges containing 60 mg of MPA. Ewes exposed to 60 mg of MPA were marked by the rams at a significantly later (P < 0.00001) mean time (54.8 ± 1.44 h) than ewes receiving 10 mg sponges (43.6 ± 1.08 h). These results reveal the potential for modifying ovulation rate through short-term glucogenic manipulations, at least during the compressed follicular phase typical of ram-induced ovulations.     

Effects of PMSG and ram contact on the reproductive performance of progestagen-treated ewes during breeding and anestrous seasons

The objective of this study was to evaluate the efficacy of combinations of PMSG treatment and ram contact on the reproductive performance of progestagen-treated ewes during three different times of the year, Febraury (early anestrus), July (late anestrus) and October (breeding season). A total of 109 multiparous Dorset ewes was used. Ewes were treated with intravaginal progestagen pessaries for 12 days, injected with 500 IU PMSG at pessary removal and either isolated from rams prior to mating (n = 12, February; n = 12, July; n = 8, October) or exposed to rams during pessary treatment (n = 17, February; n = 12, July; n = 8, October). A third treatment group (n = 18, February; n = 6, July; n = 8, October) received pessaries and ram exposure but no PMSG. An additional treatment of progestagen pessaries alone was included in October (n = 8). There were no differences among treatments in their ability to induce estrus at different times of the year, but incidence of estrus tended (P < 0.10) to be lower for PMSG treatment during the July breeding. During February, the use of pessaries with PMSG treatment increased (P < 0.05) conception and lambing rates, whereas ram contact was without any beneficial effects. The trend was reversed during July breeding, when ram contact increased (P < 0.05) fertility of progestagen-treated ewes compared with other treatment combinations. Pessaries alone were sufficient to attain acceptable levels of fertility and fecundity in October.