Evidence for maternal regulation of early conceptus growth and development in beef cattle

Fifty-one cyclic beef cows were mated with fertile bulls. At 36 h after the start of oestrus, cows were assigned to receive sesame oil (controls) or progesterone (100 mg) on Days 1, 2, 3 and 4 of pregnancy. Peripheral plasma concentration of progesterone was measured until slaughter on Days 5 or 14. Cows were randomly assigned to be slaughtered on Days 5 or 14 or remain intact and palpated per rectum on Day 40 to verify pregnancy. Uteri on Days 5 and 14 were flushed for recovery of luminal protein and conceptus tissue. Conceptus and endometrial tissues were cultured with [3H]leucine and submitted to two-dimensional-PAGE and fluorography. Administration of progesterone increased peripheral plasma progesterone concentration on Day 2-5. Conceptuses recovered from progesterone-treated cows on Day 14 were advanced in development compared to conceptuses from control cows. Conceptuses recovered from progesterone-treated cows were viable as polypeptides associated with maintenance of pregnancy in cattle were synthesized and released at an earlier time and pregnancy was maintained beyond Day 40. Early progesterone stimulation altered the synthesis and release of polypeptides from endometrial explant cultures on Day 5. Results indicate a role of progesterone in the maternal regulation of conceptus growth and development in early pregnancy of cattle.     

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of intrauterine and cervical manipulation on the equine oestrous cycle and hormone profiles.

Endometrial biopsy or endometrial biopsy and uterine culture taken on Day 4 after oestrus induced lysis of the corpus luteum (CL), resulting in a sharp decline in serum progesterone concentration and shortened the interoestrous interval in 8/12 and 32/33 oestrous cycles, respectively, during 2 experiments. Cervical dilatation 4 days after oestrus shortened the interoestrus interval in 5/10 and 0/5 oestrous cycles. Endometrial biopsy and culture on Days 1 and 3 after oestrus also induced CL lysis during 4 of 7 cycles. Total oestrogen (oestrone plus oestradiol) concentrations increased at the onset of the subsequent oestrus in mares biopsied on Day 4 of dioestrus or in control cycle oestrous periods. Endometrial biopsy also induced lysis of the CL in mares with persistent luteal function. It is postulated that intracervical or intrauterine manipulations during the luteal phase of the oestrous cycle may directly, or indirectly, stimulate the release of an endogenous luteolysin (prostaglandin) resulting in CL regression, followed by oestrus and ovulation in the mare.     

Endometrial histology and post-partum mares treated with progesterone and synthetic GnRH (AY-24,031).

Foal heat was significantly delayed in 15 Thoroughbred and Quarter-horse mares by 200 mg progesterone in oil from Days 5--14 post partum. Nine of these mares subsequently received daily i.v. injections of 2 mg of a synthetic GnRH preparation (AY-24,031) from Day 2 of the progesterone-delayed oestrus but this treatment did not significantly shorten oestrus or hasten ovulation. Uterine biopsies taken on Day 15 post partum from all the mares showed a mixed endometrial morphology having both oestrous and dioestrous characteristics. There was an increased proliferation of endometrial glands in these animals at the time of ovulation compared to control mares having a normal foal heat.     

The effect of the gonadotrophin-releasing hormone analog, buserelin, administered in diestrus on pregnancy rates and pregnancy failure in mares

Two trials involving 578 mares were performed to investigate the effect of a single intramuscular treatment of 40 microg buserelin, an analog of gonadotrophin releasing hormone, on pregnancy rate in mares. All mares were bred by natural mating and were allocated into pairs One mare in each pair was injected with buserelin either on Day 10 or 11 (Trial 1) or on Days 8 to 10 (Trial 2) after ovulation. Pregnancy status of mares was determined by transrectal ultrasonographic examination on Day 14 or 15 after the day of ovulation and was repeated between Days 28 and 30 of pregnancy. In Trial 1, buserelin treatment increased the pregnancy rate at Days 14 and 15 (72.5 vs 66.6%, P < 0.01). At the second pregnancy examination, pregnancy losses were lower in the treated group of mares (4.1 vs 7.4%; P < 0.05). In Trial 2, buserelin also improved the pregnancy rate (57.2 vs 53 5%; P < 0.05) at Days 14 and 15 Pregnancy losses between the first and second examinations were lower in the treated group of mares (6.5 vs 12.0%; P < 0.05). Buserelin increased pregnancy rates after breeding at the first estrus in both trials. In addition, buserelin treatment increased the pregnancy maintenance rate at Days 28 to 30.     

FSH and LH concentrations in periparturient mares.

The influence of the ovaries and presence of a foal on periparturient concentrations of FSH and LH were studied in 19 Pony mares. In intact and ovariectomized mares, mean concentrations of FSH fluctuated between 1.1and 9.9 ng/ml on Days -14 to-1 before parturition (Day 0). A surge of FSH occurred in all mares in association with parturition. From Days 1 to 10, the high levels of FSH gradually decreased in the intact group to the minimal concentrations that occur during oestrus, but remained elevated in the ovariectomized mares. There were no significant pre-partum changes in LH in either type of mare. Post-partum changes in LH concentrations increased at a similar rate in ovariectomized and intact mares. The presence of a foal significantly lengthened the interval to first oestrus, depressed LH levels on Days 6--10 and decreased the FSH concentrations as averaged over the 10 days before the first ovulation after parturition.     

Effect of maternal treatment with altrenogest on age at puberty, hormone concentrations, pituitary response to exogenous GnRH, oestrous cycle characteristics and fertility of fillies

Puberty was studied using 15 fillies of Quarter Horse phenotype. Fillies were from dams treated daily from Days 20 to 325 of gestation with: (1) 2 ml neobee oil per 50 kg body weight (controls); or (2) 2 ml altrenogest (2.2 mg/ml) per 50 kg body weight. The clitoris was measured at birth and approximately every 12 weeks until 84 weeks of age. Blood samples were collected from 9 fillies (5 treated, 4 controls) every 4 days over a 28-day period at 8-week intervals from 4 to 68 weeks of age; sampling continued every 4 days after 72 weeks of age until first oestrus. Blood samples were collected daily during oestrus (greater than or equal to 35 mm follicle) and on Days 4, 6, 10, and 14 after ovulation for the first 2 oestrous cycles. GnRH challenges (5 micrograms/kg) were administered every 8 weeks from 32 to 96 weeks of age. Puberty was defined as the first oestrus with ovulation. Beginning 1 February 1987, fillies were teased daily and their ovaries were examined by ultrasonography every 3 days (daily during oestrus). Fillies were inseminated with 500 x 10(6) motile spermatozoa from one stallion. Pregnancy was diagnosed by ultrasonography on Days 11, 12, 15 and every 5 days until Day 50 after ovulation. Prenatal altrenogest treatment caused clitoral enlargement (P less than 0.05) and increased serum concentrations of LH from 1 to 7 months of age. The amount of LH released in response to exogenous GnRH was greater (P less than 0.05) in treated fillies at 32, 64, and 72 weeks of age. Treated fillies had higher serum concentrations of FSH from 1 to 4 months (P less than 0.05), but FSH was lower (P less than 0.05) in treated fillies before and during first oestrus. Serum concentrations of LH and FSH peaked transiently at 10 months and LH was depressed from 64 to 88 weeks and began to rise 14 days before first oestrus. Concentrations of FSH began to decline 14 days before first oestrus. The median age at puberty was 90 weeks. Durations of oestrus, dioestrus, and the oestrous cycle were not different between groups and were similar to those for adult mares. First cycle pregnancy rates and overall rates were 100 and 82% and 100 and 91.7% for control and treated fillies, respectively (P greater than 0.05). Maternal treatment with altrenogest did alter gonadotrophin secretion before puberty, but had no effect on functional reproductive performance in fillies.     

Embryo loss following GnRH-induced ovulation in anovulatory mares

Two experiments were conducted using a 21-day GnRH analogue treatment regimen to induce ovulation in seasonally anovulatory mares. In Experiment 1, nontreated (n=20) and treated (n=83) mares were defined as having inactive ovaries (largest follicle相似文献   

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.     

Synchronization of oestus and timed insemination of mares.

Oestrus was synchronized in 116 mares by means of an i.m. injection of prostaglandin F-2 alpha (Day 0) and of fluprostenol (a PG analogue) on Day 16. Mares were then randomly divided into three groups. Group A mares (N = 30) were given 2500 i.u. hCG I.M. ON Day 20 and artificially inseminated on Day 21 without detection of oestrus. Group B mares (N = 32) were given 2500 i.u. hCG i.m. on Day 20 and inseminated on Days 21 and 23, also without oestrus detection. Group C mares (N = 54) were teased on Days 18, 19, 21, 23 and 25 and inseminated on Days 19, 21, 23 and 25 while they were in oestrus. Semen was collected by artificial vagina from 3 stallions. One-third of the mares in each group were assigned to each stallion at random. The gel-free fraction was divided equally among the mares, and used within 1 h of collection. Pregnancy rates at about 60 days of gestation were not significantly different. A high rate of synchronization of oestrus (80%) was attained within 48 h of treatment with fluprostenol.     

Effect of periovulatory prostaglandin F2alpha on pregnancy rates and luteal function in the mare.

The objective of this study was to determine whether periovulatory treatments with PGF2alpha affects the development of the CL, and whether the treatment was detrimental to the establishment of pregnancy. Reproductively sound mares were assigned randomly to one of the following treatment groups during consecutive estrus cycles: 1. 3,000 IU hCG within 24 hours before artificial insemination and 500 microg cloprostenol (PGF2alpha analogue) on Days 0, 1, and 2 after ovulation (n=8), 2. 2 mL sterile water injection within 24 hours before artificial insemination and 500 microg cloprostenol on Days 0, 1, and 2 after ovulation (n=8); 3. 3,000 IU hCG within 24 hours before artificial insemination and 500 microg cloprostenol on Day 2 after ovulation (n=8); or 4. 3,000 IU hCG within 24 hours before artificial insemination and 2 mL of sterile water on Days 0, 1, and 2 after ovulation (controls; n=8). Blood samples were collected from the jugular vein on Days 0, 1, 2, 5, 8, 11, and 14 after ovulation. Plasma progesterone concentrations were determined by the use of a solid phase 125I radioimmunoassay. All mares were examined for pregnancy by the use of transrectal ultrasonography at 14 days after ovulation. Mares in Group 1 and 2 had lower plasma progesterone concentrations at Day 2 and 5, compared to mares in the control group (P < 0.001). No difference was detected between group 1 and 2. Plasma progesterone concentrations in group 3 were similar to the control group until the day of treatment, but decreased after treatment and were significantly lower than the control group at Day 5 (P < 0.001). Plasma progesterone concentrations increased in all treatment groups after Day 5, and were comparable among all groups at Day 14 after ovulation. Cloprostenol treatment had a significant effect on pregnancy rates (P < 0.01). The pregnancy rate was 12.5% in Group 1, 25% in Group 2, 38% in Group 3, and 62.5% in Group 4. It was concluded that periovulatory treatment with PGF2alpha has a detrimental effect on early luteal function and pregnancy.     

Effects of synchronization and frequency in insemination on fertility.

Fifty-four normally cycling, non-lactating mares were given 2 injections (i.m.) of PGF-2 alpha (10 mg) 14 days apart without regard to stage of the oestrous cycle. At 19 days after the first PGF-2 alpha treatment, a single i.m. injection of either hCG (3300 i.u.) or a GnRH-analogue (500 micrograms) was administered. Each mare was inseminated with 100 X 10(6) motile spermatozoa at one of the following frequencies: once only on Day 20; every other day during oestrus or at least on Days 19 and 21; or daily during oestrus or at least on Days 19, 20, 21 and 22. Eighteen control mares received saline injections on Days 0 and 14, and were inseminated either on the 4th day of oestrus or every other day or daily beginning on the 2nd day of oestrus. More (P greater than 0.05) PGF-2 alpha treated mares displayed their 1st day of oestrus on Days 14 to 20 than control mares (80.6 versus 27.8%). During cycle 1, fewer (P greater than 0.05) treated mares became pregnant compared to controls; 38.9, 25.0 and 66.7% for PGF-2 alpha + hCG, PGF-2 alpha + GnRH-A and control mares, respectively. After three cycles, the pregnancy rates for mares inseminated every other day or daily were higher (P less than 0.05) than mares inseminated only once during oestrus (88.9 and 88.2 versus 64.7%).     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Effect of diethylstilboestrol on the relationship between LH, PMSG and progesterone during pregnancy in the mare.

Two studies were conducted to determine the relationship between LH and progesterone and between PMSG and progesterone during pregnancy in mares. In the first, samples of jugular blood were collected daily from 7 mares from the first day of oestrus until Day 28 of pregnancy, and in the second, samples were collected weekly from 14 mares from Day 35 of gestation until parturition. In an attempt to prolong secretion of progesterone from accessory corpora lutea, 7 of these 14 mares were injected with increasing doses (2--10 mg) of diethylstilboestrol (DES) between Days 84 and 142 of gestation. The remaining 7 mares received injections of vehicle. Concentrations of LH, PMSG and progesterone in serum were determined by radioimmunoassay. From the onset of oestrus until Day 4 of gestation, serum concentrations of LH and progesterone were negatively correlated (r = 0.67, P less than 0.01), but from Days 5 to 28 a positive correlation (r = 0.80, P less than 0.01) was noted. Likewise, serum concentrations of PMSG and progesterone were highly correlated between Days 35 and 196 in mares injected with DES (r = 0.72, P less than 0.01) and the vehicle (r = 0.75, P less than 0.01). Injections of DES did not influence serum concentrations of LH, PMSG or progesterone, or affect the length of gestation. It was concluded that DES does not influence the maintenance of pregnancy in the mare.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

An investigation of the uterine luminal environment of non-pregnant and pregnant pony mares.

Uterine flushings were collected from 30 non-pregnant Pony mares on Days 8, 12, 14, 16, 18 or 20 after oculation. Mares were allowed a recovery period of one oestrous cycle and were mated at the next oestrus. They were then ovario-hysterectomized on days which corresponded to the day of the oestrous cycle to which they were assigned. Uterine flushings were analysed for total recoverable protein and acid phosphatase activity. Least squares analysis indicated a status X day interaction for total protein (P less than 0.10) and acid phosphatase activity (P less than 0.005) in which the latter was higher in uterine flushings during pregnancy. Peripheral plasma oestrone and oestradiol concentrations were measured by radioimmunoassay and results indicated that plasma oestrone concentrations in pregnant and non-pregnant mares were not different, and oestradiol was lower (P less then 0.005) in the peripheral plasma during pregnancy. conceptus membranes were incubated in vitro for 120 min in a chemically defined medium. Incubation medium was then assayed to assess oestrone and oestradiol production capacilities at Days 8, 12, 14, 16, 18 and 20 of pregnancy. Conceptus membrane production of oestradios (pg/5 ml/h) increased (P less than 0.05) from Day 8 (243 pg/5 ml) to Day 20 (108 763 pg/5 ml). A similar trend, but of lower magnitude, existed for oestrone production.     

Changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares

The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n = 16) or not detected (n = 8) by Day 12. Endometrial vascularity (scored 1-4, for none to maximal, respectively) did not differ on Days 1-8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P < 0.05) on Days 12-16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P < 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P < 0.05) than scores in the opposite horn on Days 12-15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P < 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility.     

Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares

Pregnancy rates at Days 2 and 14 postovulation were determined for 15 normal mares and 15 subfertile mares. Embryonic loss rates were estimated by the difference in the Day 2 and Day 14 pregnancy rates. Mares were artificially inseminated with the pooled ejaculates from three stallions, and the embryonic vesicle was detected with ultrasonography at Days 9, 10, 12 and 14. Mares were short-cycled with prostaglandin F(2) alpha (PGF(2alpha)) and rebred to the same stallions, and the Day 2 pregnancy rates were determined by recovery of cleaved ova (embryos) from the surgically excised oviducts. Significantly more (P < 0.01) normal versus subfertile mares were pregnant at Day 14 (12 15 vs 3 15 ). There was no significant difference in the Day 2 pregnancy rate for normal versus subfertile mares (10 14 vs 11 14 ). There were no significant differences (P > 0.5) in the mean number of blastomeres per embryo or in the mean diameter of embryos recovered at Day 2 from normal or subfertile mares. The estimated embryonic loss rate was significantly lower (P < 0.01) for normal verusus subfertile mares (0 10 vs 8 11 ). Fertilization rates were similar for normal and subfertile mares; however, subfertile mares had a higher embryonic loss rate prior to Day 14 postovulation.     

Delayed follicular development and ovulation following inhibition of FSH with equine follicular fluid in the mare

In two experiments, PGF(2alpha) was given to all mares on Day 10 (ovulation = Day 0). In experiment 1, mares received either whole follicular fluid or saline i.v. every 12 hours on Days 10 to 14. Experiment 2 was similar to experiment 1, except the follicular fluid was extracted with charcoal to remove steroids. Analysis of the FSH data for Days 10 to 21 indicated an effect of treatment (P<0.08) with whole follicular fluid, but not with charcoal-extracted follicular fluid. However, there was an effect of day (P<0.05) and an interaction (P<0.01) of treatment with day for both experiments. The interaction of treatment with day seemed primarily due to a marked post-treatment increase in FSH concentrations between Days 15 and 17 for mares treated with either whole follicular fluid or charcoal-extracted follicular fluid. Analysis of the diameter of the largest follicle for Days 10 to 18 indicated a main effect of treatment (P<0.05) and day (P<0.05) and an interaction (P<0.05) of treatment with day for both experiments. The interaction of treatment with day was attributable to the inhibition of follicular growth by Day 14 for mares treated with whole follicular fluid and by Day 15 for mares treated with charcoal-extracted follicular fluid. The length of the interovulatory interval was longer (P<0.05) in the treated group than in controls for both experiments. Results indicated that equine follicular fluid contained a proteinaceous substance that suppressed circulating concentration of FSH. The inhibited follicular growth and the delay in ovulation were attributed to the reduced concentrations of circulating FSH.