Effects of clomiphene citrate on ovarian function in hypophysectomized rats

On Days 28-30 of age, hypophysectomized rats were treated with oestradiol-17 beta (0.1 mg/day) and/or clomiphene citrate (0.1 mg/day). Subsequent treatment with PMSG (10 i.u., on Day 31) and hCG (10 i.u., on Day 33) was identical for all animals. Rats were killed on Day 34. Treatment with oestradiol-17 beta alone resulted in ovulations of 45.1 +/- 5.5 oocytes/rat (mean +/- s.e.m.). There were no ovulations among animals treated with clomiphene citrate alone but treatment with oestradiol-17 beta and clomiphene citrate resulted in a significant (P less than 0.05) reduction (23.1 +/- 7.6 oocytes/rat) in ovulatory response. Similarly, ovarian weights and serum progesterone concentrations were highest in the oestradiol-17 beta-treated rats, intermediate in those given oestradiol plus clomiphene citrate and the lowest in rats receiving clomiphene citrate alone. We suggest that clomiphene citrate exerts direct ovarian antiovulatory and oestrogen-antagonist actions.     

Effects of monoclonal antibody against PMSG administered shortly after the preovulatory LH surge on time and number of ovulations in PMSG/PG-treated cows

Normally cyclic heifers received 2500 i.u. PMSG i.m. at Day 10 of the oestrous cycle and 15 mg prostaglandin (PG) i.m. 48 h later. From 30 h after PG the LH concentration in the peripheral blood was estimated every hour using a rapid RIA method which allowed the LH concentration to be known within 4 h. Monoclonal antibody against PMSG was injected in the jugular vein of 29 heifers at 4.8 h after the maximum of the preovulatory LH peak; 28 heifers were not treated with anti-PMSG (controls). Peripheral blood concentrations of PMSG, LH, progesterone and oestradiol were compared. Ovaries were collected by ovariectomy at fixed times, 22-30 h after the LH peak, and numbers were counted of small (2-10 mm), large (greater than 10 mm) and ovulated follicles, and of follicles with a stigma. In anti-PMSG-treated cows, the PMSG concentration fell sharply to non-detectable levels within 2 h of the treatment, indicating that PMSG was neutralized in these cows at the onset of final follicular maturation. In all cows, the concentration of oestradiol showed a significant decrease at about 8 h after the LH peak. After anti-PMSG treatment ovulations took place from 24 until 30 h after the LH peak, whereas in control cows follicles had already ovulated at or before 22 h and ovulations continued until 30 h. At 30 h 90% of the follicles had ovulated in anti-PMSG-treated cows vs 72% in the controls, resulting in 15 and 8 ovulations per cow respectively (P less than 0.05). Also, administration of monoclonal antibody against PMSG synchronized final follicular maturation and shortened the period of multiple ovulations. In conclusion, neutralization of PMSG shortly after the preovulatory LH peak suppresses adverse effects of PMSG on final follicular maturation, leading to an almost 2-fold increase of the ovulation rate.     

Effects of gonadotrophin on the ovary of Apodemus flavicollis in winter anoestrus

Wild-caught female Apodemus flavicollis were given daily subcutaneous injections of PMSG + HCG on two consecutive days (2xPMSG + HCG), HCG on two days (2 X HCG), PMSG + HCG until their vagina became perforate (Perforate), and PMSG + HCG until they became perforate and were left undisturbed for another five days (Perforate + 5). Untreated females served as controls. The ovaries and uteri increased in weight as a result of the treatment. The number of healthy follicles increased in "perforate" females and decreased in "2 X HCG". Luteinization of the follicles and possibly of the interstitial tissue was seen in "2 X HCG". In this group, the mean diameter of the corpora lutea increased, and it decreased in "perforate" and "perforate + 5". The mean number of corpora lutea was unaffected by the treatment. The activity of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) was strong in the corpora lutea of the untreated females and remained so throughout treatment. The interstitial tissue of the untreated females showed only weak 3 beta HSD activity, but the activity was strong in all the experimental groups. One female ovulated, but it is not clear if it was in response to treatment.     

Ovum recovery in superovulated cows and cleavage rates in the fertilized ova

Four groups of 10 cows were given one, two, three, or four thousand i.u. of PMS on Day 16 of the estrous cycle. On Day 19, the cows were injected with 10–15 mg of estrogen. At the ensuing estrus, the cows were bred and given 2,000 i.u. of HCG. The cows were slaughtered for ovum recovery 3–7 days after breeding. On the average, the cows produced 10.3 ± 1.6 ovulations with a range from 0 to 41. Each 1,000 i.u. of PMS was associated with an increase of 5.83 ± 1.18 in number of ovulations. The variation among cows within treatment groups was not attributable to differences in age or body weight. On the average, 51.5% of the ovulated ova were recovered, with the recovery rate being higher (64.2%) for ova in the oviducts and lower (34.2%) for those in the uterus. Ovarian length at the time of slaughter was directly related to PMS dosage and a smaller proportion of the ova were recovered from animals having largest ovaries. Only 38.4% of 211 recovered ova were fertilized. It was estimated that cleavages occurred at intervals of 12.6 ± 23.6 hours.     

Effects of time of PMSG and fixed-time GnRH injections on estrus incidence and fertility in physiologically different ewes pre-treated with progestogen-impregnated vaginal sponge during the nonbreeding season

A 2 × 2 factorial study for hormonal treatment was designed in 85 seasonally anestrous ewes with physiologically different status (nonparous, dry, and postpartum). All ewes were pre-treated with 60 mg of 6-methyl-17-acetoxy-progesterone (MAP) vaginal sponge for nine days and divided into four groups: Group I (22 ewes) — an i. m. injection of 600 i.u. pregnant mare's serum gonadotropin (PMSG) at the sponge removal (Day 0) and a single i.m. injection of 100 ug synthetic gonadotropin releasing hormone (GnRH) at 36 h after the sponge removal; Group II (21 ewes) — a PMSG injection at Day 0 and a saline injection at 36 h after the sponge removal; Group III (21 ewes) — a PMSG injection two days before the sponge removal and the GnRH injection at 24 h after the sponge removal; and Group IV (21 ewes) — a PMSG injection at Day -2 and a saline injection at 24 h after the sponge removal. The treated ewes were allowed to mate once with rams for five days after treatment. Estrus incidence and lambing rates were low (69.4% and 27.1%, respectively), probably due to the mating system and poor body condition of ewes used in the study. No effect of PMSG injection two days before with-drawal of MAP sponge and the fixed-time GnRH injection were observed in estrus incidence, fertility, and prolificacy. The present study indicates that the physiological status of ewes combined with management systems including feeding and mating would be important for out-of-season breeding with hormonal treatment.     

Endocrine responses of goats after induction of superovulation with PMSG and FSH

Goats in Group A were pretreated for 9 days with a synthetic progestagen, administered via intravaginal sponge, and 1000 i.u. PMSG s.c. on Day 12 of the oestrous cycle. Goats in Group B had the same PMSG treatment, but not the progestagen pretreatment. Group C goats received a s.c. twice daily injection of a porcine FSH preparation (8 mg on Day 12, 4 mg Day 13, 2 mg Day 14 and 1 mg Day 15). Oestrus was synchronized in all animals by 50 micrograms cloprostenol, 2 days after the start of gonadotrophin treatment. The vaginal progestagen sponges were removed from Group A at the same time. Mean ovulation rate was slightly higher in FSH-treated than in the PMSG-treated animals, whereas the incidence of large follicles that failed to ovulate was significantly elevated in PMSG-treated animals in Group B. More goats in Groups A and B than in Group C exhibited premature luteal failure. Progestagen pretreatment appeared to suppress both follicular and luteal activity, as indicated by numbers of large non-ovulating follicles and by the magnitude and duration of elevated plasma oestradiol levels following PMSG stimulation, and by decreased plasma progesterone levels before and after PMSG treatment. Oestrogenic response to FSH was considerably less than that to PMSG, as indicated both by a considerably shorter duration of elevation of circulating oestradiol levels during the peri-ovulatory period, and by lower maximal oestradiol levels. Differences in the ovarian responses to PMSG and FSH may be attributed primarily to differences in the biological half-life of each preparation.     

Ovarian response to exogenous hormones in six-week-old lambs.

Crossbred lambs 5--6 weeks old were treated with human chorionic gonadotrophin (hCG) (500 or 1500 i.u.) alone, hCG plus pregnant mare serum gonadotrophin (PMSG) (1000 or 2000 i.u.), 1000 i.u. PMSG alone, or were untreated. PMSG alone and PMSG + hCG increased ovarian weight and uterine weight. PMSG alone stimulated growth and luteinization of follicles but PMSG + hCG induced ovulations and formation of corpora lutea. hCG alone did not change any of the characteristics which were measured. PMSG had a significant effect on the number of vesicular follicles but none of the treatments affected the number of growing follicles.     

Ovarian function in Holstein cows immunized against pregnant mare serum gonadotrophin

Six Holstein-Friesian cows were immunized against pregnant mare serum gonadotrophin (PMSG) using Freunds' adjuvant during the mid-luteal phase of the estrous cycle. Antibody response was maintained by five booster immunizations at 2- to 3-wk intervals. Four cows were treated with a single intramuscular injection of PMSG (2350 I U) 107 d after primary immunization. Cloprostenol (500 ug) was administered at 56 h and 72 h after the treatment with PMSG; the cows were inseminated three times at 12-h intervals starting 56 h after cloprostenol treatment. Five days after insemination, the animals were slaughtered and their reproductive organs were recovered to quantify the population of corpora lutea and unovulated follicles (>10 mm dia). Antibody titres and progesterone concentrations were determined from blood samples collected either on alternate days or twice a week. Initially, progesterone concentrations were measured in milk samples. All cows produced antibodies, and titres were elevated within 6 to 9 d following each booster immunization. After each boost, however, the antibody titres declined rapidly. Progesterone concentrations declined to below 1 ng/ml after two weeks of initial immunization and remained low throughout the study, except in one cow that ovulated on Day 75. All animals were observed to have large follicular cysts during this period. Treatment with PMSG induced a single ovulation in one cow. Ovulations were neither induced by PMSG nor observed in any of the other animals. In PMSG-treated animals, the mean number of large follicles (5.0) was greater than in those which were not treated (2.0). The results of this study suggest that low titres of antibodies against PMSG are sufficient to disturb ovarian activity, result in follicular cysts and block multiple ovulations in response to exogenous PMSG.     

Ovarian response to PMSG treatment in ewes immunized against oestradiol-17 beta

The effects of active immunization against oestradiol-17 beta on the ovarian response to pregnant mare serum gonadotrophin (PMSG) was investigated in Merino ewes. Immunized (79) and control (41) ewes were synchronized with intravaginal sponges, given either 750 or 1500 i.u. PMSG and then mated to rams or inseminated laparoscopically with fresh diluted semen. All control ewes mated naturally exhibited oestrus and 40 out of 41 control ewes ovulated. The ovulation rate was higher in the controls receiving 1500 i.u. PMSG than in those ewes which received 750 i.u. PMSG (10.2 v. 3.3). Immunization against oestradiol-17 beta resulted in antibody titres varying from 100 to more than 100 000 in plasma taken 1-4 days after mating. The ovarian response increased significantly in the lowest titre group (100-1000) in conjunction with stimulation with 1500 i.u. PMSG. In these ewes the ovulation rate increased over controls (16.7 v. 10.2) as did the total ovarian response, which includes follicles greater than 10 mm diameter (22.3 v. 11.1). The total ovarian response was also increased in those ewes given 750 i.u. PMSG which had titres in the 1000-10 000 and 10 000-100 000 range, but this was not accompanied by significant increases in the ovulation rate. In general, the higher titre levels (greater than 1000) were correlated with decreases in the proportion of ewes showing oestrus and ovulating and in the embryo recovery rate. The 1500 i.u. PMSG treatment group with the highest titres (greater than 10 000) also showed a significant drop in the ovulation rate as compared to the 1500 i.u. PMSG controls.     

Interference with the preovulatory luteinizing hormone surge and blockade of ovulation in immature pregnant mare's serum gonadotropin-primed rats with the anti-androgenic drug, hydroxyflutamide

The involvement of androgens in the control of ovulation has been assessed by administration of the androgen antagonist, hydroxyflutamide, to prepubertal rats treated with pregnant mare's serum gonadotropin (PMSG) to induce first estrus and ovulation. Without human chorionic gonadotropin (hCG) injection, only 46% of rats that received six 5-mg, s.c. injections of hydroxyflutamide at 12-h intervals, beginning an hour before s.c. injection of 4 IU PMSG on Day-2 (Day 0 = the day of proestrus), had ovulated a mean of 1.3 +/- 0.4 oocytes per rat when killed on the morning of Day 1, whereas 92% of sesame oil-treated controls had ovulated a mean of 6.9 +/- 0.6 oocytes. After i.p. injection of hCG at 1600 h on Day 0, 92% of hydroxyflutamide-treated rats ovulated a mean of 8.3 +/- 1.2 oocytes compared to 100% of controls, which ovulated 7.3 +/- 0.4 oocytes per rat: these groups were not significantly different from each other, nor from control rats that received no hCG. Thus, exogenous hCG completely overcame the inhibitory effect of hydroxyflutamide on ovulation. Rats treated with PMSG and hydroxyflutamide without hCG were killed either on the morning of Day 0 to determine serum and ovarian steroid levels or on the afternoon of Day 0 to determine serum LH levels. Serum levels of estradiol-17 beta and testosterone in hydroxyflutamide-treated rats were significantly higher (178% and 75%, respectively; p less than 0.01) than levels observed in controls on the morning of Day 0. Ovarian concentrations of the steroids were also elevated in hydroxyflutamide-treated rats (p less than 0.01 for testosterone only).(ABSTRACT TRUNCATED AT 250 WORDS)     

Superovulation in cattle: A combined treatment using syncromate B with either pregnant mare serum gonadotrophin or follicle stimulating hormone

A comparison between different superovulatory treatments in dairy cattle was carried out at a commercial embryo transfer unit in Israel. Both pregnant mare serum gonadotrophin (PMSG) and follicle stimulating hormone (FSH) were used, either alone or combined with Syncromate B (SMB). The use of PMSG + SMB significantly decreased the number of corpora lutea present at the time of embryo collection 7 d after insemination, as compared with other treatment regimens. Consequently, a significantly lower number of ova was found in those animals treated with PMSG + SMB. Better superovulatory responses were obtained when FSH, rather than PMSG, was used, regardless of whether they were administered alone or combined with SMB. It was clear that the use of SMB combined either with PMSG or FSH resulted in poorer responses than when either gonadotrophin was used alone.     

Fertility, ovulation and maturation of eggs in mares injected with HCG

Pony mares were observed from January to August for incidence of oestrus, duration of oestrus, length of the oestrous cycle and for ovulation and fertility after injection of HCG. From January to 15 May most mares showed oestrus but the duration of oestrus was quite variable and few mares ovulated in response to HCG. From 15 May to 17 August oestrous cycles were more regular and ovulation was induced within 40-50 h by an intramuscular injection of 1500-5000 i.u. HCG. Pregnancy was established by one mating at a fixed time after HCG in 20 of 69 mares. Degenerate eggs were recovered from the oviducts of anoestrous recently ovulated, mated, unmated and pregnant mares. The first polar body was formed before ovulation in 2 eggs and had not formed in 2 recently ovulated eggs flushed from the oviduct. The second polar body formed after sperm penetration 10-12 h after ovulation. After formation of pronuclei, the first cleavage division occurred at 20 h and the second at 32 h after ovulation. Oestrus was inhibited by progesterone administered by vaginal devices but occurred within 1-3 days in 12 of the 20 mares after withdrawal of the devices.     

Comparison of LH release and luteal function in cyclic and LH-RH-treated anoestrous ewes pretreated with PMSG or oestrogen.

Pretreatment of seasonally anoestrous Clun Forest ewes with 750 i.u. PMSG or 50 microgram oestradiol benzoate 24 or 7 h respectively before a single injection of 150 microgram synthetic LH-RH significantly increased the release of LH compared to that after injection of 150 microgram LH-RH alone. Total LH release in the two "combined" treatments was approximately 70% of that found at a natural oestrus, compared to 25% for LH-RH alone. All but one of the treated ewes ovulated, but only those pretreated with PMSG consistently produced corpora lutea capable of elevating peripheral plasma progesterone concentrations although these were lower than those at natural mid-cycle. These progesterone concentrations were, however, comparable to those during the natural cycle when corrected for the higher metabolic clearance rate found during anoestrus.     

Effects of gonadotropins on follicular development, ovulation, and atresia in the mature guinea pig

The induction of follicular growth, ovulation, and atresia by heterologous gonadotropic preparations was studied late in the reproductive cycle of the adult female guinea pig. Human chorionic gonadotropin (HCG) administration (10 IU) 12 days following the first signs of opening of the vaginal membrane was found to stimulate ovulation within 24 h in all animals studied, as evidenced by recovery of ova from their oviducts as well as the presence of postovulatory follicles in their ovaries. Histologically, ovaries of animals receiving HCG exhibited atretic changes in most of the follicles smaller than 999 micrometer in diameter. Pregnant mares serum gonadotropin (PMSG, 10 IU) administered on days 9 and 10 of the cycle was not sufficient to stimulate ovulation in this species although histological changes in the follicular complement were observed. Administration of PMSG prior to the HCG appeared to have an inhibitory effect on ovulation induction. Follicles luteinizing with entrapped ova were seen in all groups receiving exogenous gonadotropin, although they were most prevalent in the animals receiving the maximum total gonadotropin doses (i.e. PMSG + HCG).     

Effect of suppression of plasma prolactin on ovulation, plasma gonadotrophins and corpus luteum function in LH-RH-treated anoestrous ewes.

Nineteen Scottish Blackface ewes were given LH-RH (3 X 30 micrograms i.v., 90-min intervals) during anoestrus when prolactin levels were elevated. Plasma levels of prolactin were suppressed with CB 154 (twice daily, i.m.) on Days -5 to 0 (N = 5), 0 to +5 (N = 5) or -5 to +5 (N = 5) around the day of LH-RH treatment (Day 0). Control animals (N = 4) received saline on Days -5 to +5. Nine animals ovulated forming corpora lutea as judged by laparoscopy on Day +7. No difference in FSH or LH levels was found between treatments and ovulations occurred equally in all treatment groups. Progesterone levels were less than ng/ml in all animals up to Day 14. It is concluded that short-term suppression of prolactin does not affect the incidence of ovulation or corpus luteum progesterone production in LH-RH-treated anoestrous ewes.     

Effect of gonadotrophins and testosterone on the seminiferous tubules of the immature rat.

The actions of HCG and PMSG for different periods and of testosterone of the immature rat testis were studied. Short-term administration of HCG (1-3 days) induced an early meiotic and postmeiotic stimulatory effect but a decrease in spermatogonial numbers. Administration of HCG for longer periods (10 days) caused a reduction in numbers of all cell types. Treatment with HCG + PMSG reduced the amount of inhibition, while PMSG alone resulted in histological and humoral signs of stimulation of the interstitial tissue and the meiotic and postmeiotic stages; the numbers of spermatogonia were not affected. Testosterone caused stimulation of the meiotic and postmeiotic stages and a reduced number of spermatogonia. It is concluded that while PMSG directly stimulates spermatogonia, HCG acts through testosterone secretion at the meiotic and postmeiotic stages. The early inhibitory effects of HCG and testosterone on spermatogonial numbers could be ascribed to the inhibition of endogenous FSH by androgens.     

周龄和激素水平对长爪沙鼠超数排卵效果的影响

目的探讨不同周龄和激素水平对长爪沙鼠超数排卵效果的影响,以期确定长爪沙鼠最佳超排周龄和激素使用剂量。方法腹腔注射10 IU PMSG/HCG对4～18周龄8个年龄段的雌性长爪沙鼠进行超数排卵,末次注射16～17 h内对各组动物卵母细胞计数,确定最佳超排周龄后,对该年龄动物以5、10、15 IU3个剂量水平腹腔注射PMSG/HCG,观察各组动物的卵母细胞计数差异。结果与其它周龄组相比,6周龄组长爪沙鼠超数排卵后的卵母细胞数最多,各组间有统计学意义（P〈0.05）,而5、10、15IU等3个剂量组的超排效果也有一定的差异,10 IU组数量最高。结论对长爪沙鼠而言,采用10 IU激素注射和6周龄的动物进行超数排卵,获得的卵母细胞数量最多而且超排效果稳定性。     

Comparison of control of oestrus and ovulation in sheep by an ear implant (SC-21009) or by intravaginal sponge (cronolone or map)

Oestrus and ovulation were observed in 234 Galway ewes in the breeding season in a preliminary evaluation of an implant progestagen treatment. A miniature ear implant (3 mg SC-21009) was used in a comparison with two intravaginal sponges (30 mg Cronolone and 60 mg medroxy progesterone acetate (MAP)). Each progestagen treatment was used in conjunction with pregnant mare serum gonadotrophin (PMSG) (0, 375 i.u. and 750 i.u.). The percentage of ewes showing oestrus after treatment was significantly affected by progestagen (Cronolone, 95%; MAP, 71%; SC-21009, 74%; P < 0.01), but not by PMSG dose level (0.1 < P < 0.2). Oestrus onset was similar among treatments, but heats ended significantly earlier in implant sheep. Significantly (P < 0.01) more ewes ovulated following Cronolone treatment (92%) than following MAP (74%) or SC-21009 (71%), and there was also a significant effect of PMSG (0, 73%; 375 i.u., 86%; 750 i.u., 93%; P < 0.01). PMSG dose level had a highly significant effect on the mean ovulation rate (0, 1.23; 375 i.u., 1.52; 750 i.u., 2.12; P < 0.001).     

A PMSG/GnRH method for the superovulation of the monovulatory brush-tailed possum (Trichosurus vulpecula)

Optimal ovarian stimulation with minimal degenerative changes was achieved in brush-tailed possums 3 days after a subcutaneous injection of 10 i.u. PMSG. This treatment alone did not result in ovulation and only rarely in oocyte maturation. Ovulation of 8-24 mature oocytes occurred when on the 3rd day after 10 i.u. PMSG the female received 3 intramuscular injections of 50 micrograms synthetic gonadotrophin-releasing hormone (GnRH) 90 min apart. Superovulation was achieved in immature, cycling, pregnant and lactating females, but not in animals in the preovulatory phase of a natural oestrous cycle.     

Action of PMSG on follicular populations in the heifer

The short-term action of PMSG on the population of growing follicles in cattle was studied using histological methods. On Day 7 of a synchronized oestrous cycle 10 Friesian heifers were unilaterally ovariectomized. The remaining ovary was immediately stimulated by an injection of PMSG (2000 i.u.) and was removed 48 h after the preovulatory discharge of LH. Control animals did not receive any injection of PMSG. In all ovaries, follicles greater than 70 micron diameter were counted, measured and checked for atresia. The mitotic index in granulosa cells of follicles of different sizes was estimated in both ovaries of all the PMSG-injected animals. Unilateral ovariectomy alone had no significant effect on follicular populations. In the interval between PMSG injection and removal of the second ovary (148 +/- 22.7 h), PMSG significantly increased the number of normal preantral follicles but did not change the number of normal antral follicles. The mitotic index doubled in preantral and early antral follicles but remained unchanged in large antral follicles. PMSG stimulated slightly the growth of the antrum in large antral follicles but did not stimulate its formation in preantral follicles. The incidence of atresia among antral follicles, particularly the largest ones (diam. greater than 1.7 mm), was significantly reduced after PMSG, suggesting some 'rescue' of follicles from atresia.