Number Of Large Follicles Research Articles

Effects of follicular status at treatment on follicular development and ovulation in response to fsh in spanish merino ewes

Cyclic Spanish Merino ewes were treated on Day 13 of the estrous cycle with 12 mg, im, FSH-P in saline (n = 9) or propylene glycol (n = 24), currently with 100 μg, im, Cloprostenol (Day 0). From Day -6 to Day 0, the ewes were observed daily by transrectal ultrasonography; after Day 0, ultrasonography was performed every 12 h for 72 h. Sizes and locations of ≥2mm follicles were recorded at each observation. The ovulation rate was determined by laparoscopy on Day 7 after estrus. The number of ovulations ranged from 0 to 6 in ewes treated with FSH-P in saline and from 0 to 16 in ewes receiving FSH-P in propylene glycol (P < 0.05) In the latter group, the response was bimodally distributed; about half of the females had 1 ovulation, whereas the remainder had >4 with a mean of 7 ovulations. The ovulation rate was associated with 2 characteristics of the largest follicle present at treatment (Day 0) First, if the largest follicle on Day 0 had not changed in diameter from Day -1 to Day 0, then 7 of 9 ewes had >3 ovulations; if the largest follicle had either increased or decreased, only 8 of 24 ewes had >3 ovulations (P < 0.05). Second, there was a linear trend (P < 0.07) for ovulation rate to decrease as the persistence of the largest follicle at treatment increased; no ewe in which the largest follicle on Day 0 remained present for more than 36 h ovulated more than 6 follicles. As with the ovulation rate, the numbers of large follicles on Days 1.5, 2 and 2.5 varied with the interaction of change in diameter of the largest follicle on Day 0 from Day -1 to Day 0 and with vehicle. In summary, the superovulatory response was affected by the change in diameter from Day -1 to Day 0 of the largest follicle on Day 0 and the period required for that follicle to regress after treatment with FSH-P and cloprostenol.

Effects of lighting program on reproductive development of female large white turkeys

Female commercial turkeys were used to investigate the effects of lighting program on onset of egg production, reproductive development and egg production. Under a conventional (CON) lighting program, birds were photostimulated from 5L:19D to 14L:10D at 29 wk, with an additional hour of daylight added at 35 wk of age (15L:9D). The alternate (ALT) lighting program involved weekly 0.5 h decreases in day length from 14L:10D at 3 d to 6L:18D, at 17 wk followed by weekly 0.5-h increases in day length beginning at 20 wk, to a maximum of 15L:9D at 38 wk of age. Birds were killed either at 3-d intervals (140–206 d), to assess reproductive development, or on the day following the first oviposition. A final group of birds was maintained for egg-production records. The ALT birds reached first egg at a younger age (220 d vs. 224 d) and lower body weight (11.18 kg vs. 11.88 kg) than CON birds did. The oviduct of the CON treatment reached its mature weight 5 d before the ovary. The growing oviduct of the ALT birds reached its mature weight 4 d after the ovary. Ovarian development of the ALT birds was accelerated by 10 d, compared with that of the CON birds. The number of unreconciled ovulations, as evidenced by postovulatory follicles at first oviposition, did not differ between treatments (1.2, ALT; 1.6, CON). The number of large follicles was not different between the two treatments (12.9, ALT; 14.3, CON [P = 0.092]). First-egg weights were significantly lower (70.6 g, ALT; 77.2 g, CON) for the ALT treatment, which also had a higher incidence of small eggs for the first 4 wk of production (3.31%, ALT; 0.82%, CON). Total egg production and total settable egg-production did not differ between treatments. Key words: Turkey, sexual maturation, lighting program, ovary, oviduct

Changes in follicular populations following treatment of buffaloes with PMSG (eCG) and Neutra-PMSG for superovulation

Some 19 buffaloes were synchronized by administration of a prostaglandin (PG) salt Lutalyse, with a single intramuscular (i.m.) injection of 25 mg at day -13. Luteolysis was induced by administration of 50 mg PG, in divided doses of 30 and 20 mg i.m. 12 h apart on day 0 of experiment. The 30 mg PG injection was designated as 0 h of experiment. Group I animals (n = 6) received saline and served as controls while animals in Groups II (n = 7) and III (n = 6) received 2500 I.U. PMSG (eCG) i.m. at day -2. Group III animals were administered 5 ml Neutra-eCG intravenously at 60 h. The number of follicles, classified on the basis of diameter as small (2-5 mm), medium (6-9 mm) and large (> or = 10 mm) was assessed by ultrasonography on days -2, -1, 0, 1, 2, 5 and 7 of experiment. The number of corpora lutea (CL) was recorded by palpation per rectum on day 8. The number of small follicles which did not differ among the three groups on days 0, 1 and 2 was significantly lower (P < 0.05) in Group II animals compared to those in Groups I and III on days 5 and 7. The number of medium follicles increased after eCG treatment and was significantly higher (P < 0.05) in animals of Groups II and III on days 0 and 1, compared to control animals of Group I. It was, however, not different among the three groups on subsequent days of experiment. The number of large follicles which did not differ among the three groups on days -2, 0, 1 and 2 was significantly higher in Groups II (P < 0.01) and III (P < 0.05) animals compared to those of Group I on day 5. On day 7, the number of large follicles was in the order (P < 0.05) Group II > Group III > Group I. The number of CL in Group II animals was significantly higher (P < 0.05) than that in Group I animals but was not different from that of Group III animals. These results suggest that treatment of buffaloes with eCG for superovulation reduces the number of small follicles and increases the number of large follicles 5-7 days after PG treatment. Administration of Neutra-eCG 60 h after PG treatment can partly reverse this trend but has no effect on ovulation rate. The possibility that part of the variability in ovulation rates in this study may have resulted from Neutra-eCG been given prior to or at the LH surge, or from the absence or presence of a dominant follicle at the time of eCG treatment cannot be ruled out.

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

Terminal follicular growth, ovulation rate and hormonal secretion after melatonin pretreatment prior to FGA-PMSG synchronisation in Suffolk ewes at the onset of the breeding season

The first experiment was performed in order to determine the effect of exogenous melatonin followed by a fluorogestone acetate (FGA) and pregnant mare's serum gonadotrophin (PMSG) treatment on terminal follicular dynamics, ovulation rate and luteinizing hormone (LH) pulsatility in Suffolk ewes at the beginning of the breeding season. Therefore, 10 ewes (MEL/FGA/PMSG) were injected melatonin 22 days before receiving sponges containing 40 mg of FGA for 14 days and 800 IU of PMSG at the removal of pessaries. Ten other ewes (FGA/PMSG) were synchronised with the same FGA/PMSG treatment without melatonin. The second experiment was undertaken to determine the effects of exogenous melatonin followed by a FGA-PMSG synchronisation on the plasma FSH, E 2 and P 4 concentrations. The investigations on terminal follicular growth and LH pulsatility were repeated. Six ewes were submitted to a MEL/FGA/PMSG treatment and six other to a FGA/PMSG control following the same design as in the first experiment. Melatonin treatment was effective in increasing ( p < 0.05) the circulating melatonin during a period of more than 14 days but was ineffective in lowering plasma PRL concentrations. LH pulsatility during and after the hormonal treatment and the concentrations of plasma FSH and P 4 were comparable among MEL/FGA/PMSG and FGA/PMSG groups. Concentrations of plasma E 2 during the luteal phase but not during the follicular phase of the synchronised estrous cycle were lower ( p < 0.05) in ewes treated with melatonin when compared to controls. The two groups differed only at the time of selection : the percentage of medium follicles undergoing atresia was lower ( p < 0.05) in the MEL/FGA/PMSG than in the FGA/PMSG (38% and 35.5% versus 65% and 61.5%); this resulted in a increased ( p < 0.05) mean number of large follicles (4.7 ± 0.5 versus 3.2 ± 0.5) and ovulation rate (2.6 ± 0.4 versus 1.45 ± 0.35).

Follicular dynamics during the oestrous cycle in dairy goats

AbstractDevelopment of all follicles with antral diameter ≥2 mm during the oestrous cycle was characterized in goats of the Murciana-Granadina dairy breed by daily transrectal ultrasonography. Of nine does initially synchronized in oestrus, six returned to oestrus and three did not. In the first six does, mean cycle length was 21·2 (s.e. 2-3) days. There were mean total numbers of 6·2 (s.e. 0·6) small (2 to 3 mm), 1·9 (s.e. 0·3) medium (4 to 5 mm), and 1·0 (s.e. 0·2) large follicles (≥6 mm) each day but only numbers of large follicles differed with day of cycle (P &lt; 0·01). The diameters of the largest follicle (LF1), the second largest (LF2) and the remaining follicles (RF) varied with day of cycle (P &lt; 0·01). The LF1 and LF2 varied with day in similar patterns but the difference in diameter between them (P &lt; 0·05) was greatest on days 7 and 9 (2·3 mm) and days 19 to 23 (2·2 to 3·8 mm), and least on days 2, 15, 16 and 18 (0·5 to 0·8 mm). The relationship between the LF1 and LF2 to the RF differed only early or late in the cycle when concentrations of progesterone were low (P &lt; 0.05). There was some evidence to support follicular growth in waves. Although the number of apparently new follicles detected (4·8 (s.e. 0·4) per day) did not vary with day of cycle, follicles emerging at 3 mm over a 48-h period that grew to a diameter of ≥6 mm were distributed non-randomly in three of six cyclic does. The three cystic does that did not return to oestrus by day 25 had large follicles (16 to 28 mm), fewer new follicles (3·3 (s.e. 1-1) per day; P &lt; 0·01) than cyclic does (4·8 (s.e. 0·4) per day) and fewer follicles that became largest (4·0 v. 8·8 in cyclic does; P &lt; 0·01).

Priming effect of exogenous oestradiol on luteinizing hormone secretion in prepubertal lambs.

The effect of repeated administration of oestradiol-17beta on luteinizing hormone (LH) secretion was studied in 3-month old female lambs (n = 20). Animals were randomly divided in five groups and treated or not treated (group I) with 1, 2 or 3 i.m. injections of oestradiol in corn oil vehicle (1 microg/kg). Animals were slaughtered 12 (group II) and 24 h (group III) after the first injection, 24 h after the second injection (group IV) and 24 h after the third injection (group V). Animals in groups IV and V were catheterized and blood samples were collected every 4 h starting before treatment until time of sacrifice. In the rest of the groups blood samples were taken at the time of slaughter. The number of follicles > 1 mm in diameter on the ovarian surface were recorded and classified according to size. Maximum levels of oestradiol ranged from 103 to 250 pmol/l and returned to baseline within 12 to 16 h after each injection. LH secretion showed a consistent pattern in all lambs, with increases between 8 and 16 h after each oestradiol injection. The highest amplitude and longest duration (8-12 h) of LH secretion was recorded after the second oestradiol injection. Preliminary data indicated that FSH secretion resembled that of LH. There was an increase in the number of follicles with a diameter of more than 2 mm. Plasma concentrations of progesterone and cortisol were low and did not differ within groups or between treatments. The findings confirm that the pituitary LH release system in ewe lambs is sensitive to the stimulatory effects of oestradiol long before puberty. Results indicate that priming with oestradiol increases pituitary LH release to subsequent challenges of oestradiol, but long time exposure to oestradiol may have a negative effect on LH secretion. Although none of the oestradiol-treated lambs ovulated, the increase in the number of large follicles with repeated injections of oestradiol suggests that small follicles were gonadotrophin-responsive and stimulated by the gonadotrophin release.

Effects of level of food intake on ovarian follicle number, size and steroidogenic capacity in the ewe.

The effect of high (H) or low (L) levels of food intake, during the preceding 4 weeks, on ovarian follicle numbers and steroidogenic capacity were investigated in groups of 12 adult Scottish Blackface ewes. Ewes of the two treatments had similar levels of body condition at the time of study but there was a twofold difference in levels of food intake. Ovaries were surgically removed on day 11 or 12 of the oestrous cycle (luteal phase; n = 6 per nutritional treatment) or at 30 h after injection (i.m.) of prostaglandin F2alpha analogue on day 11 or 12 of the cycle (follicular phase; n = 6 per nutritional treatment). Ovarian follicles > 1 mm diameter were dissected out and incubated individually for 2 h at 37 degrees C, in 1 ml of medium 199 which was then assayed to determine concentrations of oestradiol and testosterone. There were significantly more small follicles (1-2.5 mm diameter) in (H) than (L) ewes (P < 0.05) but no treatment difference in the numbers of large follicles (> 2.5 mm diameter) during either phase of the cycle and no difference in the mean diameters (mm) of the two largest follicles in each animal. However, although there were higher rates of synthesis of both oestrogen (P < 0.05) and testosterone (P < 0.01) in the large follicles of (L) ewes as compared with (H) ewes, there was a lower oestrogen/testosterone ratio in (L) than (H) follicles which may indicate a lower level of aromatase activity in (L) follicles. It is concluded that the effects of level of food intake on ovulation rate are expressed through differences in late stages of follicle development, probably through effects on the intrafollicular steroid milieux.

Influence of flushing on lh secretion, follicular growth and the response to estrus synchronization treatment in suckled beef cows

The effects of energy supplementation (flushing) on LH and estradiol secretion, follicular growth and the response to estrus synchronization treatment (Norgestomet+PMSG initiated 41.9±3.4 d after calving) were investigated in 16 suckled beef cows fed either 70% (Group C, n=8) of energy requirements from calving to 3 wk after AI or fed the same restricted diet until 11 d before synchronization and then were supplemented with 2 kg concentrate until 3 wk after AI (Group S, n=8). Concentrations of LH and estradiol 17 β were measured from 3 sampling periods: 25 and 39 d after calving and between 29 and 49 h after implant removal. Ovaries were examined by ultrasonography 11 d before treatment to implant withdrawal (IR). The effects of energy level, day (or hour) of observation and corresponding interactions were tested on repeated measurements by split-plot ANOVA. No positive effect of flushing was observed on characteristics of LH secretion on Day 39. However, the size of the largest follicle and the number of large follicles were higher in Group S than in Group C cows, respectively, 7 and 9 d after the beginning of flushing to 2 d after the start of treatment. After IR, the estradiol secretion tended to be higher in Group S than in Group C cows (9.8±0.4 pg/mL vs 7.2±0.2 pg/mL ; P=0.06), but no effect on LH secretion was observed. After implant removal 12 cows ovulated (Group S: 7/8 vs Group C:5/8; P>0.05), 7 were pregnant at 21 d after AI (Group S:6/8 vs Group C:l/8 ; P<0.05) and 4 at 45 d after AI (Group S: 4/8 vs Group C 0/8; P>0.05). To conclude, flushing had a positive effect on follicular growth, which does not seem to be mediated by LH. In cows fed a restricted diet, flushing enhanced follicular growth, increased the fertilization rate and/or reduced early embryonic death.

Superstimulation of ovarian follicular growth with fsh, oocyte recovery, and embryo production from zebu (bos indicus) calves: Effects of treatment with a gnrh agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140±3 kg) were randomly assigned to 3 groups: control (n=10); deslorelin treatment from Day -8 to 3 (n=10); and cetrorelix treatment from Day -3 to 2 (n=10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P<0.001) plasma LH (0.30±0.01 ng/ml) than control heifers (0.17±0.02 ng/ml), while plasma LH was reduced (P<0.05) in heifers treated with cetrorelix (0.13±0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles ≥ 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P>0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P>0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.

Gonadotropin secretion and development of ovarian follicles during oestrous cycles in heifers treated with luteinizing hormone releasing hormone antagonist

The hypothesis tested was that reduced LHRH stimulation of the anterior pituitary would lead to attenuated development of ovarian follicles as a result of reduced gonadotropin secretion during oestrous cycles of cattle. Twenty heifers were randomly assigned to be treated ( n = 5/treatment) with an antagonist to LHRH (LHRH-Ant) 1) from Day 2 to 7 (Day 0 = behavioural oestrus), 2) Day 7 to 12, 3) Day 12 to 17, 4) or serve as untreated control animals. LHRH-Ant suppressed LH pulses of heifers in all treatment groups from treatment initiation through Day 17 as compared with untreated control heifers [Peters et al., 1994. Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers. Biol. Reprod., 51 (1994) 1248-1254]. Circulating concentration of FSH from Day 8 to 10 of the oestrous cycle did not increase in heifers treated with LHRH-Ant from Day 2 to 7 or Day 7 to 12; however, there was increased (P < 0.05) FSH from Day 8 to 10 of the oestrous cycle in heifers treated with LHRH-Ant from Day 12 to 17 and control heifers. Compared with control heifers, heifers treated with LHRH-Ant from the Day 2 to 7 had suppressed (P < 0.05) size and persistence of the first and second dominant ovarian follicles. Heifers treated with LHRH-Ant from Day 7 to 12 had suppressed size (P < 0.05 and tended (P < 0.10) to have a shorter persistence of the second dominant ovarian follicle compared with control heifers. Heifers treated with LHRH-Ant from Day 12 to 17 had a similar (P > 0.10) size and persistence of dominant ovarian follicles but had reduced (P < 0.10) numbers of large follicles compared with control heifers. Heifers treated with LHRH-Ant from Day 2 to 7 had lower (P < 0.01) concentrations of 17 beta-oestradiol during the treatment period and tended (P < 0.10) to have lower concentrations of 17 beta-oestradiol from Day 7 to 12 of the oestrous cycle compared with control heifers. Heifers treated with LHRH-Ant from Day 7 to 12 or Day 12 to 17 had similar (P > 0.10) circulating LH concentrations of l7 beta-oestradiol compared with control heifers. Reduced LHRH stimulation of the pituitary from Day 2 to 12 of the oestrous cycle and the resulting modulation in circulating LH and FSH led to suppressed ovarian follicular development and oestradiol secretion. After Day 12 of the oestrous cycle, reduced LHRH stimulation of the anterior pituitary did not lead to altered ovarian follicular development to the extent as reduced LHRH stimulation before Day 12 although pulsatile release of LH was similarly suppressed by treatment with the LHRH-Ant.

Dual suppression with oral contraceptives and gonadotrophin releasing-hormone agonists improves in-vitro fertilization outcome in high responder patients.

Certain patients have a tendency for high response to gonadotrophin therapy which is often not ameliorated with prior gonadotrophin-releasing hormone agonist (GnRHa) suppression. As a result, these patients are frequently cancelled and often experience ovarian hyperstimulation syndrome (OHSS) episodes during in-vitro fertilization (IVF)-embryo transfer cycles. Patients with polycystic ovarian syndrome (PCOS) have been noted to be particularly sensitive to exogenous gonadotrophin therapy. We have developed a protocol which is effective in improving IVF outcome in high responder patients, including those with PCOS. Oral contraceptive pills (OCP) are taken for 25 days followed by s.c. leuprolide acetate, 1 mg/day, which is overlapped with the final 5 days of oral contraceptive administration. Low-dose gonadotrophin stimulation is then initiated on the third day of withdrawal bleeding in the form of either human menopausal gonadotrophins or purified urinary follicle-stimulating hormone at a dosage of 150 IU/day. Over a 5 year period, we reviewed our experience utilizing this dual method of suppression in 99 cycles obtained in 73 high responder patients. There were only 13 cancellations prior to embryo transfer (13.1%). The clinical and ongoing pregnancy rates per initiated cycle were 46.5 and 40.4% respectively. Only eight patients experienced mild-moderate OHSS following treatment. For those patients who had undergone previous IVF-embryo transfer cycles at our centre, significant improvements were noted in oocyte fertilization rates, embryo implantation rates and clinical/ongoing pregnancy rates with this protocol. Hormonal analyses revealed that the chief mechanism may be through an improved luteinizing hormone/follicle-stimulating hormone ratio following dual suppression. An additional feature of this dual method of suppression is significantly lower serum androgen concentrations, particularly dehydroepiandrosterone sulphate.

The dominant follicle exerts an interovarian inhibition on FSH-induced follicular development

An experiment was conducted to evaluate the role of the dominant follicle (DF) of the first wave in regulating follicular and ovulatory responses and embryonic yield to a superovulation regime with FSH-P. Twenty normally cycling Holstein-Freisian heifers (n = 20) were synchronized with GnRH and pgf 2α and randomly assigned to a control or a treated group (n = 10 each). Treated heifers had the first wave dominant follicle removed via transvaginal, ultrasound-guided aspiration on Day 6 after a synchronized estrus. All heifers received a total of 32 mg FSH-P given in decreasing doses at 12 h intervals from Day 8 to Day 11 plus two injections of pgf 2α (35 mg and 20 mg, respectively) on Day 10. Heifers were inseminated at 6 h and 16 h after onset of estrus. Follicular dynamics were examined daily by transrectal ultrasonography from Day 4 to estrus, once following ovulation, and at the time of embryo collection on Day 7. Blood samples were collected daily during the superovulatory treatment and at embryo collection. Follicles were classified as: small, ≤ 5 mm; medium, 6–9 mm; or large, ≥ 10 mm. Aspiration of the dominant follicle was associated with an immediate decrease in large follicles, and a linear rate increase in small follicles from Day 4 to Day 8 just prior to the FSH-P injections, (treatment > control: +0.33 vs. −0.22, number of small follicles per day; P < 0.10). During FSH-P injections, the increase in number of medium follicles was greater (P < 0.01) for treatment on Day 9–11 (treatment > control: Day 9, 3.2 > 1.8; Day 10, 9.2 > 4.7; Day 11, 13.1 > 8.3; ± 0.56). Number of large follicles was greater in treatment at Day 11 (5.12 > 1.4 ±0.21; P < 0.01). Mean number of induced ovulatory follicles (difference between number of follicles at estrus and Day 2 after estrus) was greater in treatment (13.4 > 6.3 ± 1.82; P < 0.01). Plasma estradiol at Day 11 during FSH-P treatment was greater in treatment (32.5 > 15.8 ± 2.6; P < 0.01). Plasma progesterone at embryo flushing (Day 7 after ovulation) was greater in treatment (7.4 > 4.9; P < 0.02); technical difficulties at embryo recovery reduced sensitivity of embryonic measurements. No changes in the distribution of unfertilized oocytes and embryo developmental stages were detected between control and treatment groups. Presence of dominant follicle of the first wave inhibited intraovarian follicular responses to exogenous FSH.

Changes in peripheral inhibin levels and follicular development following treatment of buffalo with PMSG and Neutra-PMSG for superovulation

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day −13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at −48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day −12 to Day −4, every 24 h from Day −4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days −2, −1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 ± 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.

GnRH improves the recovery rate and the in vitro developmental competence of oocytes obtained by transvaginal follicular aspiration from superstimulated heifers

In this study we assessed the effect of GnRH on the recovery rate, meiotic synchronization and in vitro developmental competence of oocytes recovered close to the expected time of ovulation. Twenty-three heifers were superstimulated with FSH, and luteolysis was induced by PGF 2α injection 48 h after the start of treatment Twelve heifers received 200 μg GnRH at 34 h after PGF 2α treatment, Blood samples were collected between 35 to 47 h after PGF 2α administration to determine the time of the LH surge. Transvaginal follicular aspiration was performed at 60 h after PGF 2α, and the recovered oocytes were fertilized or fixed either immediately or after 24 h of maturation in vitro. GnRH-treated heifers showed an LH surge within 3 h after treatment, while only 4 of the 10 heifers in the control group exhibited an LH surge by 47 h after treatment with PGF 2α. The average number of large follicles (> 10 mm) was 21.3 ± 2.3 and 19.3 ± 2.4 for GnRH-treated and control heifers, respectively. The oocyte recovery rate was 87.7 and 63.1% (P < 0.05), respectively, and most of the cumulus-oocyte-complexes (COC) recovered from the 2 groups had an expanded cumulus (80.4 and 80.5%, respectively). Oocytes with an expanded cumulus from the GnRH group had completed meiotic maturation at higher rate than the controls (97 vs 20%;P < 0.05). In vitro development to the blastocyst stage of cumulus-expanded oocytes fertilized immediately after recovery was higher in GnRH-treated than in control heifers (60.3 vs 40.0%; P < 0.05). No difference was observed when oocytes with compact or expanded cumulus were matured in vitro for 24 h before fertilization. These results indicate that GnRH injections improve the oocyte recovery rate and that oocytes have a higher development competence than those obtained from non-GnRH-treated animals. We propose that this higher in vitro developmental competence may result from a more synchronous or further advanced meiotic maturation. However, due to the small number of oocytes in our study, we must emphasize that our findings on meiotic resumption are of preliminary nature.

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2α im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 ± 2.4 (mean ± SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.

Ovarian response and FSH profile in cows following injection of various doses of inhibin antiserum.

Dose effect of inhibin antiserum on ovarian response and hormonal profiles were investigated. On day 12 of the estrous cycle (day 0 = estrus), 14 of 19 cows were given a single i.v. injection of 25 ml (n = 4), 37.5 ml (n = 5) or 50 ml (n = 5) antiserum against inhibin produced in a castrated male goat. The other 5 animals were given 50 ml castrated male goat serum (control serum). The animals in each group received a single i.m. injection of 0.5 mg prostaglandin F2 alpha analogue (PG) 48 hr following the serum injection. The population of follicles and ovulation rate (estimated by the number of corpora lutea) were examined by ultrasonography. Administration of inhibin antiserum consistently resulted in a significant (p < 0.01) increase in plasma concentrations of follicle-stimulating hormone (FSH) in inhibin-neutralized groups, although the increased FSH levels were sustained longer in 50-ml group than in the 25- and 37.5- ml groups. Levels in the circulating inhibin antibody titer were positively correlated with dosage of inhibin antiserum. A large number of antral follicles (> or = 4 mm in diameter) developed similarly after hypersecretion of FSH in all neutralized groups, coupled with a rise in plasma estradiol levels, while the number of large follicles (> or = 10 mm in diameter) on estrus showed a dose-dependent increase. Multiple ovulation (2 to 4) was recorded in all animals after injection of 50 ml inhibin antiserum, however all cows in the 25-ml group experienced only one ovulation and injection of 37.5 ml resulted in a variable number of ovulations (1 to 5). These results demonstrated that administration of inhibin antiserum on day 12, followed by injection of PG, was able to induce hypersecretion of FSH and subsequently multiple ovulations. The number of large follicles on estrus day and ovulations were affected by dosage of inhibin antiserum and were correlated with persistence of increased FSH levels or circulating antibody levels.

Temporal aspects of ovarian follicular growth and steroidogenesis following exogenous follicle-stimulating hormone in Angus heifers.

Ultrasonography and endocrine assay techniques were used to monitor structural and hormonal alterations made by the ovary in response to the biological actions of pituitary-derived follicle-stimulating hormone (FSH-P). Angus heifers (n = 36) were allotted to receive injections (twice per day) of either FSH-P (up to a total of 28 mg over a maximum of 4 days beginning on Day 10 of a synchronized estrous cycle) or saline in order to quantify temporal relationships among follicle growth and steroid hormone profiles. Transrectal ultrasonography was utilized at 12-h intervals to monitor and record follicle growth. Plasma was collected every 12 h for the first 48 h of the experiment and then every 6 h for the remainder of the experiment. At 48 and 60 h after the onset of treatments, prostaglandin F2 alpha (PGF2 alpha; 25 mg) was administered (i.m.). FSH-treated heifers (n = 6 at each time) were terminated at 24, 48, 72 and 96 h following the onset of treatment. Saline-treated heifers were terminated at 24 and 96 h (n = 6 at each time). After ovaries were obtained, follicular number and size were recorded and follicular fluid (FF) was collected. Plasma concentration of progesterone (P) and estradiol (E2) and FF concentration of P, E2, estrone, testosterone and androstenedione were determined by radioimmunoassays. Plasma concentration of E2 increased (P < 0.05) within 36 h of initiation of FSH treatment. Plasma P decreased (P < 0.0001) by 12 h post-PGF2 alpha. Ultrasonographic examination revealed a significant decrease in the number of small follicles by 48 h, whereas the number of medium follicles increased (P < 0.05) by 60 h after the initiation of FSH treatment. The number of large follicles (LF > or = 10 mm diameter) increased (P < 0.01) over the course of the experiment. The total number of ovarian follicles (TF) 24 h after the start of FSH treatment was correlated (r = 0.99; P < 0.0001) with the number of small follicles (SF < or = 5 mm). At 72 h after the onset of FSH treatment, the number of medium follicles (i.e. 6-9 mm) was correlated with TF (r = 0.97; P < 0.0001). Estradiol was the predominant FF steroid. Follicular fluid E2 was greatest in follicles at 72 h after FSH treatment. Follicular fluid E2 and plasma E2 were positively correlated (r = 0.66; P < 0.001). Follicular aromatase activity was estimated by evaluating the ratio of FF estrogens (E) to androgens (A). Elevated aromatase activity (E:A ratio > 1.0) was detected in 196 of 206 follicles. The estrogen to progesterone ratio was used as an estimate of follicle viability. Eighty-five percent of the follicles were estimated to be viable (E:P ratio > 1.0). The peak E:A ratio in LF preceded by 24 h the peak concentration in FF E2 and plasma E2. In MF and SF the E:A ratio increased by 72 h. Enhancement of ovarian follicular growth (i.e. increased number and size of follicles; increased steroidogenesis) by exogenous, pituitary-derived FSH is characterized by (1) increased activity of aromatase, and (2) accumulation of FF E2, events which temporally preceded the increase in plasma concentration of E2. These observations will aid efforts to incorporate recombinant bovine FSH and somatotropin in an effort to develop more predictable superstimulation and ovulation induction protocols.

Follicular 3 beta-hydroxysteroid dehydrogenase and cytochromes P450 17 alpha-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation.

In this study, we tested the hypothesis that there is altered abundance of transcripts of genes coding for the enzymes cytochrome P450 17 alpha-hydroxylase (P450(17 alpha)), cytochrome P450 aromatase (P450arom), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in follicles of cattle hyperstimulated with eCG compared to FSH. Treatments were initiated on Day 10 of the cycle, and all cows received prostaglandin (PG) on Day 12. In experiment 1, blood samples were taken to determine plasma progesterone and estradiol concentrations during ovarian stimulation. In experiment 2, both ovaries were removed from stimulated cows by colpotomy before (n = 4 cows/treatment) and at 12 (n = 3/treatment) and 24 h (n = 3/treatment) after PG injection, and from nonstimulated controls (n = 4) 72 h after PG. The preovulatory follicle from nonstimulated heifers, and all follicles greater than 3 mm in diameter from superovulated heifers, were isolated and classified as small (3-5 mm), medium (6-9 mm), or large (> 9 mm). Steady-state levels of RNA for 3 beta-HSD, P450(17 alpha), and P450arom genes were determined by Northern analysis in the individual follicles. In experiment 1, stimulation with eCG significantly (p < 0.01) increased plasma progesterone concentrations compared to FSH-stimulated and nonstimulated controls, and increased (p < 0.05) plasma estradiol concentrations compared to FSH-stimulated controls. Stimulation with FSH did not alter progesterone concentrations, but significantly increased plasma estradiol concentrations compared to those of controls. In experiment 2, the number of large follicles increased significantly with time (p < 0.01), but there were no differences between eCG and FSH treatments in size distribution of follicles (p > 0.05). Relative abundance of P450(17 alpha) message (per 20 micrograms RNA) was significantly higher in large and small follicles (p < 0.05) in eCG-treated compared to FSH-treated heifers after PG injection. Analysis within this period revealed significant treatment effects at 12 h but not 24 h after PG injection. The bovine P450arom cDNA hybridized to 3 transcripts: a 6.5-kilobase (kb) polyadenylated transcript, and non-polyadenylated messages of 3.4 and 1.8 kilobases (kb), all of which hybridized with an oligonucleotide probe specific for the heme-binding region. In medium and small follicles, the 6.5-kb and 3.4-kb transcripts were present in similar quantities, and the 1.8-kb transcript was 25% less abundant. In large follicles recovered after luteolysis, the 3.4 and 1.8-kb transcripts were 3- to 4-fold more abundant in eCG-treated compared with FSH-treated and nonstimulated animals (p < 0.05). There were no significant differences between eCG and FSH treatments on steady-state 3 beta-HSD mRNA levels. Levels of 3 beta-HSD and P450(17 alpha) mRNA in large follicles in hyperstimulated heifers were not different from those in preovulatory follicles in nonstimulated cows. We conclude that hyperstimulation with eCG results in greater stimulation of follicular P450(17 alpha) message abundance compared to hyperstimulation with FSH, and that this may contribute to increased follicular estradiol secretion.

Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo.

Bovine follicular fluid was aspirated from follicles of 2-20 mm in diameter, charcoal-treated to remove steroids and then separated into low and high molecular mass fractions. The low molecular mass (< 10 kDa) fraction was purified on a Sephadex G-25 chromatography column with formic acid as the eluent. Seven peaks were isolated and assayed for biological activity in cultures of bovine granulosa cells at concentrations of 10, 100 and 1000 ng ml-1. One peak (peak 4) inhibited (P < 0.001) the proliferation of granulosa cells when measured by cell counting and by [3H]thymidine incorporation (33-37% inhibition). This peak inhibited proliferation of granulosa cells from both small (< 2 mm) and medium (2-10 mm) follicles, but not large (> 10 mm) follicles. The inhibitory effect of peak 4 was not due to a toxic effect on cells. Administration of peak 4 to rats did not affect liver or kidney masses but did decrease uterine (25%, P < 0.01) and ovarian (35%, P < 0.01) masses. Peak 4 also caused a reduction in the number of large follicles (65%, P < 0.01) but increased the number of small follicles (55%, P < 0.01). We have named the inhibitory factor associated with peak 4, granulosa cell-inhibitory factor (GCIF). The results presented suggest that GCIF may be a factor secreted by dominant follicles that inhibits the development of subordinate follicles.