Superovulation in heifers given FSH initiated either at day 2 or day 10 of the estrous cycle

The aim of this study was to determine if initiation of superovulation in heifers during the time of development of the first dominant follicle (Days 2 to 6) would give equivalent ovulation and embryo production rates as treatment initiated at mid-cycle. Estrus was synchronized in 60 beef heifers using luprostiol (PG) and they were randomly allocated to treatment with 4.5, 3.5, 2.5 and 1.5 mg of porcine follicle stimulating hormone (FSH) administered twice daily, either on Days 2, 4, 5 and 6 (Day-2 group), respectively, or with similar doses at four consecutive days during mid-cycle (Day-10 group, initiation on Day 9 to 11). All heifers received 500 mug cloprostenol at the fifth FSH injection and 250 mug at the sixth injection. Blood samples for progesterone determination were collected at the time of FSH injections. Heifers were slaughtered 7 d post estrus, and the number of ovulations and large follicles (>/=10mm) were determined on visual inspection of the ovary. Following flushing of the uterine horns the quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using a two-sided t-test, and frequency distributions were compared using Chi-square tests. The mean number (+/-SEM) of ovulations for heifers in the Day-10 group was 12.9+/-1.0, and 8.5+/-0.9 embryos were recovered. Both the number of ovulations (6.7+/-0.8) and embryos recovered (4.1+/-0.6) were lower (P=0.0001) in heifers in the Day-2 group. Following grading based on a morphological basis, a higher number (P=0.002) of embryos was categorized as Grades 1 and 2 (4.1+/-0.6) and Grade 3 (2.1+/-0.4) in Day-10 heifers than in the Day-2 group (Grade 1 and 2, 1.9+/-0.3; Grade 3, 0.7+/-0.2). The number of Grade 4 and 5 embryos (Day 10, 1.6+/-0.2; Day 2, 1.4+/-0.2) and the number of unfertilized ova (Day 10, 0.7+/-0.4; Day 2, 0.2+/-0.1) did not differ between treatments. Progesterone concentrations were lower (P=0.0001) in Day-2 heifers at FSH treatment prior to prostaglandin, and the decline was more rapid following prostaglandin injection at Day 5 (P=0.02). Results of this study indicate that the number of ovulations and embryos recovered was lower in heifers when FSH treatment was initiated on Day 2 compared with Day 10 of the estrous cycle.     

Superovulation of dairy and beef cows using porcine FSH with defined LH content

The results of the superovulation of dairy and beef cows using porcine pituitary FSH characterized by defined LH content are reported.

A total amount of FSH equivalent to 31 mg of Armour Standard and containing LH equivalent to 500 i.u. (HMG Standard), administered in 10 decreasing doses over a period of five days, induced 7.33 ± 4.67 (mean ± SD) ovulations in six lactating Friesian cows (group 1), and 2 ± 1.41 transferable embryos were collected nonsurgically.

Furthermore, the treatment with FSH equivalent to 62 mg of Armour Standard and containing 1000 i.u. LH induced 19.43 ± 9.25 ovulations in 16 lactating Friesian cows (group 2).

Similar results were obtained in seven Marchigiana and Chianina cows (group 3) using a total amount of FSH equivalent to 46.5 mg Armour Standard and containing 750 i.u. LH.

At the higher dose, 10.56 ± 6.39 transferable embryos were collected, their percentage was 73.47%, and none of the donors produced fewer than four transferable embryos.     

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.     

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus = Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2alpha treatment on Days 6 and 7. However, the proportion of cows with > 2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with > 2 ovulations (31 vs 20%), ovulation rate. (26.0 +/- 6.3 vs 49.6 +/- 25.8), production of ova/embryos (13.3 +/- 3.2 vs 14.4 +/- 3.4), or the number of transferable embryos (8.0 +/- 3.6 vs 5.4 +/- 1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0 +/- 2.0 mm) was smaller (P<0.05) than at midcyle (16.8 +/- 1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.     

Development of persistent ovarian follicles during synchronization of estrus influences the superovulatory response to FSH treatment in cattle

The synchronization of estrus with synthetic progestins or progesterone (P(4)) results in the development of a large, persistent ovarian follicle. The objectives of the present study were to determine if development of a persistent ovarian follicle during synchronization of estrus suppresses recruitment of additional follicles during FSH treatment. On Day 5 of the estrous cycle (estrus = Day 0), beef cows were treated with 0.5 or 2.0 P(4) releasing intravaginal devices (PRIDs) for 8 d (Experiment 1, n = 20), 5 or 2 d (Experiment 2, n = 44) before initiation of FSH treatment. Prostaglandin F(2alpha) (25 mg) was administered on Days 5 and 6. Superovulation was induced with 24 mg of recombinant bovine FSH (rbFSH, Experiment 1) or 28 mg of FSH-P (Experiment 2) over a 3- or 4-d period, respectively. The PRIDs were removed concurrently with the 5th injection of rbFSH or FSH-P. There was a treatment-by-day interaction (P < 0.001) for the concentration of 17beta-estradiol in cows treated for 8, 5 or 2 d before FSH treatment. In Experiment 1, FSH treatment initiated 8 d after insertion of a 0.5 PRID did not affect the number of CL (6.9 +/- 1.4 vs 6.7 +/- 1.6), ova/embryos (3.7 +/-1.3 vs 3.0 +/- 1.3) and transferable embryos (2.4 +/- 0.9 vs 3.0 +/- 0.9) compared with that of the 2.0 PRIDs. In Experiment 2, FSH treatment initiated 5 d after insertion of a 0.5 PRID decreased the number of CL (4.0 +/- 0.5 vs 8.3 +/- 0.8; P < 0.001), ova/embryos (3.0 +/- 0.6 vs 5.9 +/- 1.2; P < 0.03) and transferable embryos (2.3 +/- 0.6 vs 5.1 +/- 1.0; P < 0.03) compared with that of a 2.0 PRID, respectively. Initiation of FSH treatment 2 d after insertion of a 0.5 PRID compared with a 2.0 PRID had no affect on the number of CL (8.0 +/- 2.1 vs 8.7 +/- 1.2), total ova (4.8 +/- 1.4 vs 6.9 +/- 1.4) and transferable embryos (2.9 +/- 1.2 vs 6.1 +/- 1.7). In conclusion, treatment with low doses of P(4) (0.5 PRID) for 5 d but not for 2 or 8 d before initiation of FSH treatment results in the development of a dominant ovarian follicle, which reduces recruitment of ovarian follicles, and the number of CL, total ova and transferable embryos.     

Use of a GnRH agonist to prevent the endogenous LH surge and injection of exogenous LH to induce ovulation in heifers superstimulated with FSH: a new model for superovulation

A new protocol for superovulating cattle which allows for control of the timing of ovulation after superstimulation with FSH was developed. The preovulatory LH surge was blocked with the GnRH agonist deslorelin, and ovulation was induced by injection of LH. In Experiment 1, heifers (3-yr-old) were assigned to a control group (Group 1A, n = 4) or a group with deslorelin implants (Group 1B, n = 5). On Day -7, heifers in Group 1A received a progestagen CIDR-B((R))device, while heifers in Group 1B received a CIDR-B((R))device + deslorelin implants. Both groups were superstimulated with twice daily injections of FSH (Folltropin((R))-V): Day 0, 40 mg (80 mg total dose on Day 0); Day 1, 30 mg; Day 2, 20 mg; Day 3, 10 mg. On Day 2, heifers were given PGF (a.m.) and CIDR-B((R)) devices were removed (p.m.). Three heifers in Group 1A had a LH surge and ovulated, whereas neither of these events occurred in Group 1B (with deslorelin implants) heifers. In Experiment 2, heifers (3-yr-old) were assigned to 1 of 4 equal groups (n = 6). On Day -7, heifers in Group 2A received a norgestomet implant, while heifers in Groups 2B, 2C and 2D received norgestomet + deslorelin implants. Heifers were superstimulated with FSH starting on Day 0 as in Experiment 1. On Day 2, heifers were given PGF (a.m.) and norgestomet implants were removed (p.m.). Heifers in Groups 2B to 2D were given 25 mg LH (Lutropin((R))): Group 2B, Day 4 (a.m.); Group 2C, Day 4 (p.m.); Group 2D, Day 5 (a.m.). Heifers in Group 2A were inseminated at estrus and 12 and 24 h later, while heifers in Groups 2B to 2D were inseminated at the time of respective LH injection and 12 and 24 h later. Injection of LH induced ovulation in heifers in Groups 2B to 2D. Heifers in Group 2C had similar total ova and embryos (15.2 +/- 1.4) as heifers in Group 2A (11.0 +/- 2.8) but greater (P < 0.05) numbers than heifers in Group 2B (7.0 +/- 2.3) and Group 2D (6.3 +/- 2.0). The number of transferable embryos was similar for heifers in Group 2A (5.8 +/- 1.8) and Group 2C (7.3 +/- 2.1) but lower (P < 0.05) for heifers in Group 2B (1.2 +/- 0.8) and Group 2D (1.3 +/- 1.0). The new GnRH agonist-LH protocol does not require observation of estrus, and induces ovulation in superstimulated heifers that would not have an endogenous LH surge.     

Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle

One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2alpha (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 microg FSH was administered (eight injections i.m. for 4 days; first on Day 13, decreasing doses) and PGFalpha analog (750 microg i.m.: Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Artificial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sanofi, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2alpha (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, < 10 and > or = 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45.5 +/- 1.4 versus 31.9 +/- 0.7; 42.0 +/- 1.6 versus 31.0 +/- 1.5; P < 0.05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade I embryos were significantly increased (P < 0.01) when animals had an interval from LH peak to first AI > or = 10 h (7.2 +/- 0.9 and 3.5 +/- 0.6) when compared to shorter intervals (4.2 +/- 1.1 and 2.0 +/- 0.7) whereas total number of embryos was unchanged (11.8 +/- 1.4 versus 10.3 +/- 1.8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is associated with a lower embryo quality when first AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time.     

Ovulations in rat ovaries perfused in vitro with follicle-stimulating hormone

Using the model of the isolated perfused rat ovary, we have found that highly purified ovine follicle-stimulating hormone (FSH) preparations cause ovulation and that this effect is not due to luteinizing hormone (LH) contamination. Ovine FSH-13 at a concentration of 1.5 mU/ml induced ovulations in all perfused ovaries (8.8 +/- 2.3 ovulations/ovary), as did a more purified preparation, ovine FSH-211B, at concentrations of 0.5 mU/ml (15.0 +/- 6.4 ovulations/ovary) and 5 mU/ml (11.3 +/- 2.6 ovulations/ovary). This ovulation-inducing effect of FSH is accompanied by a marked stimulation of estradiol levels in the perfusion medium without stimulation of progesterone levels. Furthermore, a purified rat FSH preparation (15 mU/ml) also induced ovulation in all ovaries (13.8 +/- 2.2 ovulations/ovary) as well as a stimulation of both estradiol and progesterone in the medium. These data clearly confirm the direct ovulatory effect of FSH on the ovary.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

17beta-estradiol regulation of human growth hormone (hGH), insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) axis in hypoestrogenic, hypergonadotropic women

OBJECTIVE: Ovarian hormonal function may be as important contributing factor to hGH-IGF-I-IGFBP-3 axis as age. AIM: To examine plasma hGH, IGF-1 and IGFBP-3 levels in women with premature ovarian failure compared to healthy normal controls and postmenopausal ones. PATIENTS: Group A-15 women with premature ovarian failure (POF) (mean: age 38.9+/-5.2 years, FSH 101.4+/-29.0 IU/l; 17beta-estradiol 22.5+/-14.6 ng/l). Group B consisted of 15 menopausal women (mean: age 54.7+/-2.7 years; FSH 81.9+/-32.1 IU/l; 17beta-estradiol 17.1+/- 8.0 ng/l). Group C - controls - 15 normally menstruating women (mean: age 37.1+/-9.0 years; FSH 6.2+/-1.0 IU/l; 17beta-estradiol 144.8+/-117.1 ng/l). METHODS: Body mass and BMI were measured. Basic fasting plasma hGH, IGF-I, IGFBP-3, insulin, testosterone and LH as well as prolactin (PRL), FSH and estradiol were assessed by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney u-test, Spearman rang correlation coefficient, stepwise multiple regression. RESULTS: Mean serum IGF-I level was the lowest (p<0.005) in group B (172.0+/-54.6 microg/l) and the highest in group C (273.6+/-109.0 microg/l). The mean plasma IGF-I level in group A was similar (NS) (208.3+/-66.5 microg/l) to that found in group B and lower (p<0.02) compared with that in group C. The lowest (p<0.005) serum IGFBP-3 level was found in group B (3.1+/-0.7 microg/l) compared to group C (4.4+/-0.3 microg/l). The mean plasma IGFBP-3 level (3.1+/-1.0 microg/l) in group A was lower than in group C (p<0.005) but identical as in group B. No statistically significant differences between groups were observed in mean hGH levels. Women in group A and C were younger (p<0.001) than those in group B. The lowest mean estradiol level was found in groups A and B. The highest was in group C (p<0.001). Mean plasma LH and FSH levels were higher (p<0.001) in groups A and B vs group C. In group C there were links between IGF-I and age (r=-0.60; p=0.014) The IGF-I/age relation disappeared in the groups A and B (rA=-0.26; rB=0.10; NS). The same regards IGFBP-3/ age link (rA=-0.44, NS; rB=0,31;NS). Estradiol level was related to hGH levels in group C (r=-0.54; p<0.05). In none of groups hGH/IGF-1 as well as IGFBP-3/hGH relations were found. Prolactin accounted for 69% of the variance in IGF-I level in the group B (p=0.003) and for 24% in group A (NS). Testosterone accounted for 88% (p=0.004) of the variance in IGF-I level in group B and IGFBP-3 was responsible for 86% (p=0.038) of the variance in IGF-I level in group C. Again IGFBP-3 was responsible for 47% (p=0.023) in group A and for 49% (p=0.04) in group B of the hGH variance. CONCLUSIONS: 17b-estradiol may be as important contributor to insulin-like growth factor-I (IGF-I) plasma level as age in hypoestrogenic, hypogonadotropic women.     

Changes in plasma inhibin levels following pulsatile gonadotrophin-releasing hormone therapy in a man with idiopathic hypogonadotrophic hypogonadism

Changes in circulating inhibin levels were related to changes in testosterone (T) and the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a hypogonadotrophic hypogonadal man before and during pulsatile gonadotrophin-releasing hormone therapy which resulted in normal spermatogenesis. Before treatment, the plasma inhibin levels in the patient (210 +/- 50 U/l; mean +/- SD of four samples) were lower than in normal controls (552 +/- 150 U/l; p less than 0.01), as were T (1.1 nmol/l) and gonadotrophin (less than 1.0 IU/l) levels. Within 1 week of gonadotrophin-releasing hormone treatment, plasma LH (14.1 +/- 0.7 IU/l) and FSH (14.4 +/- 0.6 IU/l) reached supraphysiological levels. In response, T and inhibin concentrations increased progressively to reach high normal levels (27.7 +/- 1.6 nmol/l and 609 +/- 140 U/l) at 4 weeks, by which time the gonadotrophin levels stared to decline and gradually returned to the normal range between 12 and 24 weeks of treatment. There was a concomitant decrease in T and inhibin levels which remained within the normal range. The decline in the FSH level following the rise in testicular hormones was earlier and steeper than that of LH (37.5% decrease at 4 weeks vs. 30.4% at 12 weeks), suggesting that T and inhibin may act together to inhibit pituitary FSH secretion as opposed to LH secretion which is primarily controlled by T. It is concluded that, in man, during maturation of the pituitary-testicular axis, changes in circulating inhibin parallel those of T, and quantitatively normal inhibin secretion is dependent on gonadotrophin stimulation. FSH secretion may be regulated through negative feedback control, by both T and inhibin.     

Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.     

Collection and transfer of multiple embryos in the mare

A pituitary extract was used to induce multiple ovulations in mares to determine whether day-7 embryos from multiple ovulators were viable as indicated by their ability to develop when transferred to recipients. There were more ovulations/donor for induced multiple-ovulating mares than for control single-ovulating mares (4.6 +/- 0.5 vs 1.0 +/- 0.0; n=14). The embryo collection rate per ovulation was similar for multiple ovulators (0.6 +/- 0.1 embryos/ovulation) and single ovulators (0.7 +/- 0.1). The embryo collection rate per donor, therefore, was higher (P<0.01) for the multiple ovulators (2.9 +/- 0.7 vs 0.7 +/- 0.1). The transfer success rate per embryo at day 21 was different (P<0.05) among recipients which received an embryo from control single-ovulating donors (7 8 ), multiple ovulators from which a single embryo was recovered (2 2 ), and multiple ovulators from which multiple embryos were recovered (9 19 ). The recipient pregnancy rate/donor at day 21 was 88% (7 8 ) for single-ovulating controls and 138% (11 8 ) for induced multiple ovulators. Results indicate that the survivability of day-7 embryos from multiple-ovulating donors was reduced. However, despite the reduced survival rate/embryo, the number of pregnant recipients/donor was increased by induction of multiple ovulations because of the increased number of embryos available for transfer.     

Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH

This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH 40 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P((R)). This compound appeared to contain 8.5 IU LH 40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (P<0.05) more corpora lutea (CL; 12.6+/-1.1) and fertilized ova (5.1+/-1.3) compared with Groups 2 and 4 (10.1+/-0.9 and 2.6+/-0.6, 9.0+/-0.9 and 2.7+/-0.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH 40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis.     

Characterization of the periovulatory period in superovulated heifers

Heifers (n=31) were superovulated with an FSH-P/cloprostenol regimen, and at 12 and 24 hours after the onset of estrus they were inseminated. Blood sampling for LH analyses and ultrasound scanning of the ovaries were performed at 4-hours intervals. The scanning, at which the first and last ovulations were recorded, was performed at 22.7 +/- 1.5 (mean +/- SD) and 31.0 +/- 1.5 hours after the LH peak, respectively. An average of 7.8 +/- 1.0 ovulations was monitored when the first ovulations were detected, while 2.8 +/- 0.7 ovulations occurred later. At 16 hours after detection of the first ovulations the oviducts were flushed and 5.6 +/- 0.5 fertilized and 2.3 +/- 0.3 unfertilized ova were isolated per animal. The fertilized ova displayed spherical pronuclei of synchronous development, and polyspermic penetration was not seen. At 24 hours after detection of the first ovulations the content of the remaining 3.3 +/- 0.5 nonovulatory follicles > 8 mm per animal was aspirated. Expanded cumulus investment was found in 69.4% of the oocytes, while 22.4% had abstricted the first polar body.     

Superovulation of goats with purified pFSH supplemented with defined amounts of pLH

The superovulatory response of goats treated with purified pFSH supplemented with 30, 40 or 50% pLH was compared. Sixty-four Boer goat does were synchronized by progestagen-containing ear implant, randomly allotted to 3 groups and, beginning 2 d before implant removal, treated with purified pFSH supplemented with 30, 40 or 50% pLH. Each animal received 16 Armour Units of pFSH administered in 6 descending doses at 12-h intervals. Along with the last 2 injections, the does received 5 mg PGF(2alpha). Embryos were flushed either surgically or after slaughter on Day 5 or 6 after the last day of standing estrus. The percentage of animals responding to treatment was not different among groups treated with pFSH supplemented with 30, 40 or 50% pLH (76, 71 and 63%, respectively). The corresponding data for number of ovulations was 11.3 +/- 1.6, 16.3 +/- 1.8 and 16.4 +/- 2.6, for number of ova and embryos recovered 8.1 +/- 1.9, 12.0 +/- 1.5 and 13.5 +/- 2.9 and for number of transferable embryos 6.6 +/- 1.9, 9.1 +/- 1.5 and 7.1 +/- 2.1 (x +/- SEM). Results confirm the earlier finding of a good response of goats to pFSH preparations with a high FSH:LH ratio, and, although group differences were statistically nonsignificant (P > 0.05), they suggest that supplementation with approximately 40% pLH may be close to the optimum.     

Effect of follicular status on superovulatory response in ewes is influenced by presence of corpus luteum at first FSH dose

The present study was developed to assess possible effects on ovulatory response and embryo yields arising from the presence of a corpus luteum (CL) at the time of initiation of the progestagen treatment used in superovulatory protocols in sheep. In breeding season, estrus was synchronized in 25 Manchega ewes using 40 mg FGA sponges for 14 days, together with a single dose of 125 microg of cloprostenol on Day 12, with Day 0 as day of progestagen insertion. Superovulatory treatment consisted of eight decreasing doses (1.5 x 3 ml, 1.25 x 2 ml, and 1 x 3 ml) of Ovagen twice daily from 60 h before to 24 h after sponge removal. The presence or absence of corpora lutea was assessed by transrectal ultrasonography at progestagen insertion and at first FSH dose. Number and size of all follicles > or = 2 mm were also evaluated at first FSH dose. The number of corpora lutea and the number and viability of recovered embryos in response to the treatment were evaluated 7 days after sponge removal. No significant effect on ovarian response of the presence of a CL at sponge insertion in 21 of the 25 ewes (84%) was detected. However, ewes with a CL at first FSH dose (16 ewes, 64%) yielded a higher number of transferable embryos (7.2 +/- 1.4 versus 2.7 +/- 0.7, P < 0.05), since the embryo degeneration rate was increased in sheep without a CL (42.5% versus 12.7%, P < 0.01). Analysis of possible effects derived from the presence of a large presumptively dominant follicle (> or = 6 mm) at first FSH dose showed that both recovery and viability rates were lowest (P < 0.05) in ewes bearing a large follicle in the absence of a CL (40.5 and 50.6%, respectively), and highest in ewes that did not show a large follicle but in which a CL was present (73.9 and 85.2%). The final number of transferable embryos was very different between groups (10.2 versus 1.8, P < 0.01). These results indicate that the number and quality of embryos obtained from superovulated ewes is affected by the presence of a CL prior to the first FSH dose (i.e. by the stage of the estrous cycle at progestagen insertion) and also by an interaction with suppressive effects from large dominant follicles. This finding suggests the existence of some effects on follicular population prior to the FSH treatment that may compromise follicle and oocyte developmental competence. It seems reasonable to hypothesize that superovulatory yields would be increased by beginning the treatment during the early-luteal phase of the estrous cycle, allowing for the presence of a CL along with the progestagen treatment.     

Progesterone is a mediator in the ovulatory process of the in vitro-perfused rat ovary

The role of steroids in the ovulatory process of the rat was explored in an in vitro perfusion system. Immature rat ovaries were primed with pregnant mare's serum gonadotropin (20 IU) and perfused in a recirculating perfusion system for up to 20 h. Unstimulated ovaries did not ovulate whereas the addition of luteinizing hormone (LH; 0.1 micrograms/ml) plus 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) resulted in 13.6 +/- 1.0 ovulations per treated ovary. Addition of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (Compound A; 10 micrograms/ml) significantly (p less than 0.01) decreased the number of ovulations after LH plus IBMX stimulation (1.6 +/- 0.8 ovulations per treated ovary). This inhibition was reversed by the addition of progesterone, with 6.6 +/- 2.1 ovulations at approximately 100 ng/ml progesterone in the perfusion medium and 15.2 +/- 3.4 ovulations at approximately 3000 ng/ml progesterone. The addition of testosterone (10 micrograms/ml) did not reverse the inhibition of ovulations by Compound A. High levels of progesterone in the perfusion medium (greater than 3000 ng/ml) did not significantly (p greater than 0.05) increase the number of ovulations after stimulation with LH plus IBMX (20.2 +/- 4.8 ovulations), and progesterone (greater than 3000 ng/ml) was not by itself able to induce ovulations. Addition of LH plus IBMX resulted in a marked increase in the levels of progesterone, testosterone, and estradiol in the perfusion medium. The production of these steroids was almost completely inhibited by the addition of Compound A, and the levels of testosterone and estradiol were restored by the addition of high concentrations of progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of the relative role of FSH and LH in the mechanism of ovulatory follicle selection in sheep

The GnRH-antagonist suppression-ovarian autotransplant model (n = 18) was used to examine the relative roles of temporal changes in FSH and LH stimulation on follicle development and selection. Follicle development was stimulated by infusion with oFSH for 3 days and treatments applied for 60 h after progestagen sponge withdrawal and before delivery of an ovulatory stimulus. In Expt 1, there was continuous infusion of FSH with or without small amplitude high frequency LH pulses, or withdrawal of FSH with or without pulsatile LH. In Expt 2, there was acute or gradual withdrawal of FSH at sponge withdrawal with pulsatile LH. The patterns of follicle development and basal and pulsatile ovarian hormone secretion were determined. The maintenance of FSH throughout the artificial follicular phase resulted in multiple follicle development and ovulation (3.3 +/- 0.3). Pulsatile LH stimulated steroid secretion (P < 0.001) but had little effect on ovulation rates (3.8 +/- 0.8) when FSH was maintained. However, withdrawal of FSH in the absence of LH resulted in atresia of the ovulatory follicles and anovulation whereas, when FSH was withdrawn in the presence of LH, preovulatory follicle development was maintained in some animals (3/6 and 5/9 in Expts 1 and 2, respectively) and these ewes had lower (P < 0.05) ovulation rates (1-2 ovulations per ewe). When FSH was withdrawn gradually in the presence of pulsatile LH, 9/9 animals ovulated with ovulation rates in the normal range. These results indicate that ovulatory follicles can transfer their gonadotrophic dependence from FSH to LH. It is hypothesized that the ability of a follicle to respond to this switch in gonadotrophic support is central to the mechanism of follicle selection.     

Serum follicle stimulating hormone, luteinizing hormone, and testosterone in sexually stimulated intact and unilaterally castrated rams

Ten two-year-old intact (IN) and unilaterally castrated (UC) Targhee rams were exposed to an estrogenized ewe each week from June to October. Each week the rams were subjectively evaluated for libido (10 for high interest and 1 for no interest). Semen was collected from all cooperating rams and evaluated for volume, concentration, and motility. Every 2 wk, blood samples were obtained at -30 and 0 min before and 30 and 60 min after ewe access. Serum was harvested; follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were quantified by radioimmunoassay (RIA). Week 5 of ewe access was assigned as Week 1. Libido scores rose from a low on Week 1, with eight rams ejaculating, to a high on Week 12, with all rams ejaculating (Week 1, 5.0 +/- 1.0; Week 12, 10.0 +/- 0.0). The product of testis length and width was significantly greater in UC compared with IN rams (88.4 +/- 1.4 versus 73.2 +/- 1.0 cm(2), respectively). Serum FSH concentrations (ng/ml) were greater (P < 0.05) in UC than IN rams and dropped over the experimental period. Serum LH concentrations (ng/ml) were significantly greater in UC compared with IN rams. This difference was more pronounced in Weeks 1 and 3 compared with Weeks 11 and 13. Serum testosterone concentrations (ng/ml) were similar in UC and IN rams throughout the experiment. In conclusion, serum testosterone was not altered in UC rams; however, serum FSH and LH concentrations were increased in UC rams. Unilateral castration did not enhance the normal changes in semen quantity and quality in the rams from July to October.