Influence of estradiol on the secretion of oxytocin and prostaglandin F2 alpha during luteolysis in the ewe.

Twenty ewes of mixed breeds were randomly assigned in equal numbers to one of four groups in a 2 x 2 factorial design. The factors were x-irradiation to destroy ovarian follicles or sham irradiation and the administration of estradiol-containing or empty (placebo) implants. Surgery for irradiation was performed on Day 8 of the cycle. Blood samples were withdrawn from jugular catheters at 1.5-h intervals from Day 10 to Day 17. Luteolysis was not observed by Day 17 in 4 of 5 placebo-treated ewes after destruction of ovarian follicles. Luteolysis was observed in 4 of 5 ewes of the sham-irradiated, placebo-treated group and in all ewes that received estradiol whether or not ovarian follicles had been destroyed. The longest (p less than 0.07) interval between peaks of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) was observed in the x-irradiated, placebo-treated group, whereas the administration of estradiol reduced (p less than 0.01) the interval between PGFM peaks. These findings indicate that a short interpulse interval in the secretion of prostaglandin F2 alpha (PGF2 alpha) is associated with luteolysis. It is possible that the reduced interpulse interval was either an effect of estradiol that caused luteolysis or a secondary event resulting from luteolysis. The administration of estradiol decreased (p less than 0.05) the number of episodes of oxytocin secretion during luteolysis and increased (p less than 0.01) the interval between episodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle

Prostaglandins, produced from membrane phospholipids by the action of phospholipase A2, cyclooxygenase, and specific prostaglandin synthases, are important regulators of ovulation, luteolysis, implantation, and parturition in reproductive tissues. Destruction of the corpus luteum at the end of the estrous cycle in nonpregnant animals is brought about by the pulsatile secretion of prostaglandin F(2alpha) (PGF(2alpha)) from the endometrium. It has been known for many years that progesterone, estradiol, and oxytocin are the hormones responsible for luteolysis. To achieve luteolysis, two independent processes have to be coordinated; the first is an increase in the prostaglandin synthetic capability of the endometrium and the second is an increase in oxytocin receptor number. Although progesterone and estradiol can modulate the expression of the enzymes involved in prostaglandin synthesis, the primary reason for the initiation of luteolysis is the increase in oxytocin receptor on the endometrial epithelial cells. Results of many in vivo studies have shown that progesterone and estradiol are required for luteolysis, but it is still not fully understood exactly how these steroid hormones act. The purpose of this article is to review the recent data related to how progesterone and estradiol could regulate (initiate and then turn off) the uterine pulsatile secretion of PGF(2alpha) observed at luteolysis.     

The non-luteolytic effect of prostaglandin F2α at the beginning of the bovine oestrous cycle: The role of oestrogen?

After the preovulatory gonadotrophin surge, antral follicles ovulate or become atretic; whatever their evolution, they stop secreting oestradiol. Since it was demonstrated that oestrogens were necessary for luteolysis to occur after PGF(2)alpha treatment, their absence could explain the non-luteolytic effect of PGF(2)alpha injected early in the cycle. Thus, cyclic cows received a PGF(2)alpha analogue and oestradiol valerate together on day 3. This treatment did not affect the life span of the corpus luteum. The absence of oestrogens in the blood does not explain the failure of PGF(2)alpha to cause luteolysis in young corpora lutea.     

Effect of treatment with estradiol valerate on endocrine changes and ovarian follicle populations in dairy cows

A linear-array ultrasound instrument was used to monitor the dynamics of follicular cyst formation following estradiol valerate (EV) administration in postpartum dairy cattle. Twelve cyclic cows were given two intramuscular (i.m.) injections of prostaglandin and F(2alpha) (PGF(2alpha)) 12 d apart to synchronize estrus. On Day 16 (Day 0 = day of estrus) six cows received 10 mg of EV in 1 ml sesame oil; the remaining six cows were treated with 1 ml sesame oil. The ovaries of all cows were scanned rectally each morning from Day 9 until 14 or 30 d post treatment. Plasma concentrations of luteinizing hormone (LH) and progesterone (P(4)) were also determined as objective indices of treatment effects. Day 0 to 16 ultrasound pictures of the ovaries of both control and treated cows were characterized by the presence of a corpus luteum (CL; 19 to 38 mm), several small follicles (<5 mm) and a medium-sized follicle (6 to 28 mm). Following treatment in control cows, the CL regressed gradually, and a preovulatory follicle was identifiable by Day 17 to 18, it increased in size and reached a maximum of 28 to 30 mm by Day 20 after ovulation and was identifiable throughout the rest of the cycle. Administration of 0 mg of EV resulted in a rapid reduction in the size of the CL. Growth of a large follicle was observed in all treated animals around Days 16 to 20, but having reached a maximum diameter of 12 to 24 mm it regressed without resulting in ovulation. Subsequent ultrasound pictures of EV-treated cows were characterized by the absence of a new CL and the presence of medium-sized persistent follicles. Estradiol valerate treatment induced early luteolysis (43 +/- 05 h post EV vs 101 +/- 22 h) and an LH surge (41 +/- 11 h vs 125 +/- 17 h).     

Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles

Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F(2)alpha 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol>/=100 ng/ml and oxytocin<65 pg/ml (preovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol<100 ng/ml and oxytocin>/=65 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol<100 ng/ml and oxytocin<65 pg/ml (atretic follicles). Treatment with prostaglandin F(2)alpha significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol: progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF(2)alpha was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles.     

Interrelationship between plasma estradiol concentration and oxytocin-induced PGF2alpha release in heifers

The objective of this study was to determine if the increase in responsiveness to oxytocin toward the time of luteolysis was correlated with an increase in plasma estradiol in the cow. Six heifers each had a cannula placed in the jugular vein on Day 14 of the estrous cycle. Then, beginning on Day 15, growth of the largest follicles was determined by ultrasonography, and a blood sample was taken via the cannula for the measurement of progesterone and estradiol by radioimmunoassay (RIA). After the first blood sample, 3 more samples were taken at 10-min intervals, 100 IU oxytocin were injected into the vein, and a further 3 blood samples were taken at 15, 30 and 60 min after injection. The concentration of 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in these frequent samplings and was used to determine the ability of oxytocin to stimulate PGF2alpha release from the uterus. This procedure was repeated daily for at least 7 d. The results showed that the response to oxytocin increased before luteolysis and that there was a significant increase in the response to oxytocin (P<0.05) before any changes in plasma estradiol or progesterone were detected. These data show that an increase in estradiol secretion from the ovulatory follicle does not appear to initiate luteolysis.     

An intra-corpus luteum site for the luteolytic action of prostaglandin F2 alpha in the rhesus monkey

It has not been possible to demonstrate prostaglandin F2 alpha (PGF2 alpha) participation in primate luteolysis under conditions of systemic administration or of acute intraluteal injection. These study designs were hampered by the short biological half-life in the first instance and brevity of administration in the latter. In this study, luteolysis has resulted from chronic, intraluteal delivery of PGF2 alpha. Using the Alzet osmotic pump-cannula system, normally cycling rhesus monkeys were continuously infused, until menses occurred, with PGF2 alpha (10 ng/1/hr) directly into the corpus luteum (CL, n = 6), into the stroma of the ovary not bearing the corpus luteum (NCL, n = 3), or subcutaneously (SC, n = 5). An additional 5 monkeys received vehicle (V) into the corpus luteum. All experiments commenced 5-7 days after the preovulatory estradiol surge. Luteal function was assessed by the daily measurements of plasma progesterone, estradiol, and LH. Intraluteal PGF2 alpha caused premature functional luteolysis in all monkeys, as reflected by a highly significant decline in circulating progesterone and estradiol and the early onset of menstruation, when compared to the other groups. V, NCL, and SC infusions had no effect on either circulating steroid levels or luteal phase lengths. None of the experimental groups showed any change in plasma LH concentrations. These are the first data to indicate that PGF2 alpha can induce functional luteolysis in the primate, and the site of action appears to be the corpus luteum.     

Destruction of bovine ovarian follicles: effects on the pulsatile release of luteinizing hormone and prostaglandin F2 alpha-induced luteal regression

Destruction of ovarian follicles during diestrus prolongs the lifespan of corpora lutea in cows, but the site(s) of action is unclear. Thus, ovarian follicles were destroyed in 10 beifers (X-IRRAD) on Day 9 postestrus, while 10 additional beifers (SHAM) served as a control group. To investigate changes in luteotropic support resulting from destruction of ovarian follicles, pulses of luteinizing hormone (LH) were characterized on Days 8, 13, and 15 postestrus. To study the interaction between products from ovarian follicles and prostaglandin F2 alpha (PGF2 alpha) in luteolysis, changes in serum concentrations of progesterone were monitored after an injection of saline or PGF2 alpha on Day 14 postestrus. Frequency and amplitude of pulses of LH increased by Day 13 in X-IRRAD beifers. An increase of similar magnitude in amplitude but not frequency of pulses of LH occurred between Day 13 and Day 15 postestrus in SHAM beifers. Exogenous PGF2 alpha was significantly less efficacious in causing luteolysis in X-IRRAD animals. We suggest that increased luteotropic support may be involved in but is not the only cause for lengthening the lifespan of corpora lutea following destruction of ovarian follicles. Additionally, we suggest that regression of bovine corpora lutea involves a synergistic action between products from ovarian follicles and PGF2 alpha.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

The role of luteal oxytocin in episodic secretion of prostaglandin F2alpha at luteolysis in the ewe

The role of luteal oxytocin in the generation of luteolytic episodes of prostaglandin F2alpha at luteolysis was investigated. On day 10 of the cycle Dorset ewes underwent either surgical removal of the corpora lutea (lutectomy; n = 4) or sham operation (sham; n = 4). Lutectomised ewes were then administered progesterone by twice daily i.m. injection in corn oil (20 mg/day) until day 14 when treatment was ceased to simulate luteolysis. The concentration of 13, 14 dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in peripheral blood samples collected at 20-min intervals for 8 h on days 12-16 of the cycle. Progesterone and oestradiol concentrations were similar in the two groups over the whole experimental cycle while oxytocin fell dramatically following lutectomy. No prostaglandin F2alpha release episodes were seen on day 12 or 13, while from days 14-16 both groups exhibited a similar episode frequency (lutectomy 0.9/ewe/8 h; sham 0.8/ewe/8 h). Analysis of episode characteristics revealed lower episode amplitude (p<0.05) but longer episode duration (p<0.05) in the lutectomy group. The results demonstrate that a normal frequency of prostaglandin F2alpha release episodes occurs independently of luteal oxytocin secretion. However, luteal oxytocin is involved in regulating the pattern of release, perhaps causing the release of episodes of the magnitude required for the successful completion of luteolysis.     

Evidence for a role of prostaglandins in the antepartum release of relaxin in the pregnant rat

The relationship of the antepartum elevation in serum relaxin levels in pregnant rats to luteolysis was examined by determining the effects of the luteolysin prostaglandin F2 alpha (PGF2 alpha) and the prostaglandin synthetase inhibitor indomethacin on antepartum serum relaxin levels, as well as on luteolysis and birth. Intravenous administration of PGF2 alpha on the morning of Day 20 elevated serum relaxin levels approximately fourfold within 15 min. Administration of the prostaglandin synthetase inhibitor indomethacin from Day 19 until Day 23 protracted luteolysis, delayed or prevented birth, and delayed the antepartum elevation of serum relaxin levels, until after indomethacin treatment had been terminated. Collectively, these results indicate that prostaglandins, in particular PGF2 alpha, may promote the antepartum increase in serum relaxin levels, as well as luteolysis and birth in rats.     

Will the first dominant follicle of the estrous cycle of heifers ovulate following luteolysis on day 7?

The pattern of turnover of dominant follicles involves the sequential growth and regression of two to three dominant follicles during the estrous cycle. The dominant follicle that ovulates is the one that develops concomitantly with the regression of the corpus luteum. The aim of this paper was to determine if the first dominant follicle would ovulate following induction of luteolysis with prostaglandin F2 alpha analogues (PGF) on Day 7 of the cycle. Heifers (n = 43) were checked for estrus (Day 0); their ovaries were scanned daily from Day 6 of the cycle for one week, and the fate of the first dominant follicle was determined. Luteolysis was induced on Day 7 with PGF analogues, and blood samples were taken daily for progesterone and estradiol measurement and at 3-h intervals for 33 h for luteinizing hormone (LH) measurement. Of the 43 heifers given PGF, complete luteolysis occurred in 40 animals. Of these, the first dominant follicle ovulated in 37 heifers; the dominant follicle was not the ovulatory follicle in 2 heifers and the dominant follicle became cystic in one heifer.     

Failure of the prostaglandin F2 alpha analogue, cloprostenol, to induce functional luteolysis in the olive baboon (Papio cynocephalus anubis)

The prostaglandin F2 alpha analogue, cloprostenol, which is an effective luteolytic agent in the common marmoset, was administered intramuscularly to olive baboons to determine if it possessed luteolytic properties in this species. The results showed that functional luteolysis was not induced when cloprostenol was administered during the mid- to late luteal phase or during early pregnancy.     

Luteal regression in the primate: different forms of cell death during naturaland gonadotropin-releasing hormone antagonist or prostaglandin analogue-induced luteolysis

Morphological changes in the corpus luteum following natural and induced luteolysis in the marmoset were investigated by light and electron microscopy. Functional corpora lutea were studied in the mid and late luteal phase, naturally regressed corpora lutea in the early and late follicular phase, and corpora lutea induced to regress by administration of GnRH antagonist or prostaglandin F(2alpha) analogue in the midluteal phase. Natural luteolysis was associated with lutein cell atrophy, condensation of cytoplasmic inclusions and organelles, and accumulation of lipid. GnRH antagonist treatment resulted in aggregations of smooth membranes and myelin-like bodies in the cytoplasm of the lutein cells together with complex aggregations of degenerative cells. After prostaglandin treatment, the lutein cells contained numerous small and large vesicles; as the degenerative changes advanced, these vesicles coalesced into alveolar-type vacuoles, and nuclei involuted. These results show that in the marmoset, natural luteolysis and the two luteolytic treatments reveal different forms of luteal degeneration and cell death, none of which fit the ultrastructural criteria for apoptosis. More emphasis needs to be placed on understanding these predominant nonapoptotic forms of cell death in order to elucidate the process of luteolysis in the primate.     

The failure of a prostaglandin F2 alpha analogue, cloprostenol, to initiate lactation in cattle

An experiment was designed to determine if the analogue of prostaglandin F2 alpha, cloprostenol, at a dose sufficient to cause luteolysis, was lactogenic in cattle. The mammary glands of eight Friesian heifers were developed by treatment with progesterone plus oestrogen. Lactation was then initiated by administration of cloprostenol and subsequent milk production was compared to that of heifers lactating after a normal pregnancy. Injection of cloprostenol failed to initiate lactation. The level of prolactin but not cortisol in blood was substantially elevated following treatment. The results cast further doubt on the importance of prolactin in the lactogenic process but indicate the likely involvement of glucocorticoids.     

Effect of sodium cloprostenol and flunixin meglumine on luteolysis and the timing of birth in bitches.

At birth, the physiological role of prostaglandins in bitches is unclear. Bitches were treated before parturition with either saline, the prostaglandin analogue, sodium cloprostenol, or the prostaglandin synthetase inhibitor, flunixin meglumine. The animals were examined regularly to determine the onset of parturition and a series of blood samples were taken to define the hormonal profiles before, during and after birth. Animals treated with cloprostenol whelped earlier than did controls. In addition, the prostaglandin F2 alpha metabolite surge and decrease in plasma progesterone concentration and rectal temperature were earlier than in controls. Flunixin meglumine disrupted the normal 13,14-dihydro-15-keto prostaglandin F2 alpha profile but did not abolish prostaglandin synthesis completely or delay the onset of labour in treated animals. This study confirms that prostaglandins induce luteolysis and the onset of labour in the bitch. However, the partial inhibition of prostaglandin synthesis does not prevent parturition.     

Immunohistochemical localization of PGF2 alpha receptor in the rat ovary.

As a step towards understanding the role of prostaglandin F2 alpha (PGF2 alpha) in ovarian function, a rabbit antiserum against purified PGF2 alpha receptor (PGF2 alpha-R) was produced. This report details the use of this antiserum in immunohistochemical staining of ovaries of non-pregnant and pregnant rats to ascertain which cell types, in vivo, possess PGF2 alpha-R. In non-pregnant rats, three ovarian cell subpopulations contain immunoreactive PGF2 alpha-R. These include: a subpopulation of the cells found in corpora lutea, a subpopulation of the thecal cells surrounding secondary and mature (Graafian) follicles, and a subpopulation of primary and secondary interstitial cells. The ovarian tissues and cell types in which immunoreactive PGF2 alpha-R cannot be demonstrated include: the serosa overlying the ovary and its vessels, the coelomic epithelium and its underlying cortical stroma, medullary stroma and vessels, granulosa cells of primary, secondary and mature follicles, the oocyte, and the blood vessels and stroma within corpora lutea. PGF2 alpha-R immunohistochemical staining of corpora lutea from non-pregnant animals was examined both prior to the start of luteolysis and during luteolysis. During luteolysis, cells undergoing apoptosis stained for the presence of PGF2 alpha-R. PGF2 alpha-R immunohistochemical staining was also examined in corpora lutea during pregnancy and until 4 days postpartum. The major findings here were the apparent large increase in staining intensity of granulosa-lutein cells during pregnancy, and the loss of PGF2 alpha-R immunopositivity of the granulosa-lutein cells during the postpartum period. In summary, three ovarian cell subpopulations, all of which can secrete steroids, possess immunoreactive PGF2 alpha-R.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of FSH, follicular development and estrus synchronization in the mare with steroid-free follicular fluid

Twenty-two pony mares were used in a project designed to determine the effectiveness of different treatments in controlling FSH, follicular development and synchronization of estrus and ovulation. Mares in Group 1 (n=8) received daily oral altrenogest (0.044 mg/kg); those in Group 2 (n=7) received daily altrenogest (0.044 g/kg) and, during the last 4 days of treatment they received steroid-free follicular fluid, (15 cc) intravenously (I.V.) two times a day; Mares in Group 3 (n=7) received daily intramuscular (I.M.) injections of progesterone (80 mg) and estradiol valerate (7 mg). All treatments lasted for 10 days, at the end of which prostaglandin (PgF(2)alpha, 10 mg) was administered. Sexual behavior, follicular development and FSH concentrations were monitor daily. Concentrations of FSH in Group 2 mares, were not significantly different (P>0.05) from those of Group 1 until the mares in Group 2 were treated with follicular fluid (P<0.05). Concentrations of FSH in Group 3 mares, were significantly lower than those of Groups 1 and 2 (P<0.05) until the mares in Group 2 were treated with steroid-free follicular fluid. At this point there was no significant difference between groups 2 and 3 (P>0.05). Steroid-free follicular fluid appears to induce atresia in larger follicles (>11 mm), and the initiation of new follicular wave. The combination of progesterone and estradiol valerate appears to delay follicular growth and not to induce atresia, since larger follicles (>11 mm) continued to grow after treatment. Both treatments (groups 2 and 3) resulted in ovulations within 5 days period. The treatment in Group 1 did not have any effect on FSH or follicular development and ovulations were dispersed through a 9-day period. We concluded that steroid-free follicular fluid offers a new possibility to synchronize ovulation in the mare by controlling FSH and follicular development.