FSH Injection Research Articles

Embryo production from superovulated cattle following insemination of sexed sperm

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 × 10 6, 10 × 10 6 or 20 × 10 6 total sperm enriched for the X- or Y-chromosome (∼90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 × 10 6 total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70–72 h following PGF 2α. Ova/embryos were collected non-surgically 7–7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 × 10 6 sexed sperm compared to higher doses ( P < 0.05). However, insemination of 20 × 10 6 total sexed sperm of ≥90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.

Power Doppler ultrasound assessment of ovarian perifollicular blood flow in women with polycystic ovaries and normal ovaries during in vitro fertilization treatment

To evaluate whether ovarian perifollicular blood flow (PFBF) varies by ultrasound among women with polycystic and normal ovaries undergoing in vitro fertilization (IVF). Prospective observational cohort study of women undergoing IVF treatment. Department of reproductive medicine at a university teaching hospital. Thirty four women with regular spontaneous ovulatory menstrual cycles undergoing IVF divided into two groups according to findings on a baseline transvaginal ultrasound scan: group 1 consisted of 20 women with ultrasound-evident normal ovaries (USNO group), and group 2 consisted of 14 women with ultrasound-evident polycystic ovaries (USPCO group). Serial transvaginal power Doppler ultrasound assessments throughout the follicular phase of ovarian stimulation. Ovarian PFBF and ovarian stromal artery pulsatility index. Women with USPCO had a significantly lower ovarian stromal artery pulsatility index at the time of the first ultrasound assessment before starting the FSH injections compared with USNO women. However, there was no difference in ovarian PFBF between women with USPCO and USNO during the follicular phase of ovarian stimulation for IVF. There is no difference in ovarian follicular vascularity between women with polycystic and normal ovaries during ovarian stimulation at IVF treatment.

326 OPTIMIZING FREQUENCY OF FSH APPLICATION FOR SUPEROVULATORY TREATMENT IN CATTLE

The aim of the study was to examine the influence of the application frequency of Pluset® (Calier, Spain) and Folltropin® (Vetrepharm, London, Ontario, Canada) on the results of embryo recovery in Simmental cattle. Moreover, pulsatility of LH concentrations in plasma should be studied before and after application of Pluset. FSH was given in a treatment of eight or four injections on four consecutive days starting between Days 9 and 13 of the estrous cycle. Luteolysis was induced by application of 500 μg Estrumate® (Essex Tierarznei, Munich, Germany), applied 72 and 84 h after the first FSH injection. Embryo recovery was performed non-surgically on Day 7 of the following estrous cycle. Heifers received either a total amount of 500 IU Pluset according to the International Standard for human urine FSH and LH (P1: 8 injections, n = 20; P2: 4 injections, n = 54) or 200 mg Folltropin according to NIH bFSH-B1 (F1: 8 injections, n = 117; F2: 4 injections, n = 60). Cows received either 550 IU Pluset (P3: 8 injections, n = 54; P4: 4 injections, n = 183) or 260 mg Folltropin (F3: 8 injections, n = 36; F4: 4 injections, n = 159). Altogether 8 heifers were bled throughout 8 h every 10 min on Days 9 and 12 (before and during stimulation with FSH). The samples were analyzed for LH by ECLIA (intra-assay coefficient of variation (VK) 6.4%, inter-assay VK 8.9%). The evaluation of the data was carried out with the procedure ‘GLM’ of the statistics software package SAS (SAS Institute, Inc., Cary, NC, USA) and PULSAR analysis. As a post hoc test Student's t-test was used. Significance was set at P = 0.05. Results of embryo recovery are shown in the table. Number of LH pulses in 8 h (LSM ± SE) was 2.3 ± 0.4 on Day 9 and 0.6 ± 0.4 on Day 12 (P &lt; 0.05). We conclude that four injections of both FSH products can lead to results comparable with those reached with eight injections in a 12 hours interval. Therefore, the effort for superovulatory treatments can be reduced. However, it has to be considered that the portion of transferable embryos was reduced, partly significantly, after four FSH injections. This result was independent of the drug used. Table 1. Results of embryo recovery

Gonadotrophins – filled-by-mass versus filled-by-bioassay

Application of recombinant technology to FSH in the early 1990s produced a highly purified preparation. Calibration of the product was dependent on the rat ‘bioassay’ based on measurement of ovarian weight after injection of FSH into rats. This test is associated with variability in the measurement of FSH bioactivity (10–20%) and in the batch-to-batch FSH content. To improve calibration of the final product, a new concept of measuring the final product by mass was developed for follitropin alfa, which has a very consistent isoform profile and a high specific activity. The new formulation of follitropin alfa (FbM) displays a very low batch-to-batch variability (<2%). The advantages of the FbM formulation have been assessed in clinical practice. A significant improvement in the consistency of the ovarian response reported in IVF cycles is likely to be related to the more precise dosing of FbM batches. Similarly, in patients treated for ovulation induction, the cycle cancellation rate was significantly reduced, as well as the need for dose adjustment and repetitive monitoring. Furthermore, the duration of treatment and the total dose of FSH required for cycles with and without IVF were significantly reduced. In conclusion, the new filled-by-mass formulation of follitropin alfa, by providing a more precise dosing of the FSH content in each batch, markedly improves the convenience and effectiveness of stimulation cycles.

57 INVESTIGATION OF CYNOMOLGUS MONKEY (MACACA FASCICULARIS) FETUS FIBROBLAST CELL NUCLEAR TRANSFER

Use of nuclear transfer (NT) in the cynomolgus monkey to establish tailor-made ES cells with the final goal of cloned embryo production was investigated. Activation stimulus conditions previously confirmed in parthenote production were used. Injection method NT was conducted using cynomolgus monkey fetus fibroblast cells in order to investigated the time it takes, from injection to activation, to reprogram the donor nuclei. Oocytes were collected under laparoscopic observation from mature cynomolgus monkeys 40 h after hCG (400 IU/kg) administration 9 days after follicle stimulation by i.m. injection of FSH (25 IU/kg). Donor cells, 40-day-old fetus fibroblast cells, were cultivated and synchronized at G0/G1 phase. After mature (MII) oocytes were enucleated using a Piezo-drive unit, donor cells were injected. At 2 h (Experiment 1, E1) and 4 h (Experiment 2, E2) after donor cell injection, activation was carried out by 2-min treatment with ionomycin and cultivation by 6-dimethylaminopurine for 4 h. As a control, parthenote production was carried out under the same activation conditions as NT. After activation, in vitro culture was carried out for about 9 days under conditions of 38°C, 5% CO2, and 5% O2. Whole-mount specimens of NT embryos were made immediately post-injection, 2 and 4 h post-injection, and 2 h after activation. Pronuclear formation (PN) and cleavage rates of NT embryos were 82.1% and 95.7% for E1, and 53.8% and 92.8% for E2, respectively. Control PN and cleavage rates were each 100%. Subsequent embryo development arrested at the 6-cell stage (8.7%) in E1 and 5-cell stage (7.1%) in E2 but proceeded to blastocyst stage (27.3%) in the control. For whole mount specimens, donor nuclei caused premature chromosome condensation in enucleated oocyte cytoplasm, and decondensation due to activation was seen, so injected donor nuclei reconstruction had occurred. No difference was seen between E2 and E1 embryo development and whole mount specimens, but E1 PN rate was clearly higher than that of E2. So 2 h of reprogramming time is more appropriate than 4 h. In this study, most NT embryos arrested at the 4-cell stage. These results suggest that development did not proceed beyond MET (maternal-embryonic transition) which is believed to occur between the 4- and 8-cell stage in cynomolgus monkey. Further study will be necessary to find the condition that completely reprograms injected donor nuclei for cloned embryo production. Table 1. Development of cynomolgus monkey fibroblast nuclear transfer embryos This work was supported by grants from the ministry of Education, Science, Sports, and Culture (13358014, 14380382).

337 OVUM PICK UP IN NON-PREGNANT AND POSTPARTUM SWAMP BUFFALOES (BUBALUS BUBALIS) AFTER FSH PRETREATMENT

The feasibility of OPU has already been reported in prepubertal swamp buffaloes and heifers (Promdireg et al. 2000 Thai J. Vet. Med. 30(1), 41–50; Techakumphu et al. 2003 Theriogenology 61, 1705–1711) and it has to be emphasized that the reproductive potential of females can be maximized, if OPU and IVEP are applied in the course of non productive periods such as anestrus and postpartum. The objective of this study was to evaluate the efficiency of Ovum Pick Up (OPU) in non lactating, pluriparous non-pregnant (n = 5) and lactating, postpartum swamp buffaloes, started 3 mts after calving (n = 6) with gonadotropin stimulation. OPU was performed every two weeks in the two groups of animals for a total of 6 sessions. To buffaloes receiving hormonal stimulation, a total of 400 mg of FSH was administered for three days in decreasing dose together with 100 μg of GnRH 24 h after the last FSH injection. The number of aspirated follicles between non-pregnant and postpartum buffaloes was not significantly different, 7.2 ± 3.7 (217) and 9.0 ± 3.2 (285), respectively (P &gt; 0.05). Recovered oocytes between the two groups of hormonally stimulated animals was also not statistically different: 3.7 ± 2.7 (112) in the non pregnant and 5.9 ± 3.5 (198) in the postpartum group, respectively (P &gt; 0.05). Among stimulated buffaloes, most aspirated follicles were of the small size (&lt;5 mm). Overall oocyte recovery rate in both the groups was 61.8% (41–77.4%). Collectively the majority of recovered oocytes were single- and multi-layered, at a higher rate in non-pregnant than postpartum buffaloes. In conclusion, as already reported in cattle, this study confirms the possibility of retrieving oocytes by OPU from non-pregnant and postpartum buffaloes. The number and quality of recovered oocytes was similar in both groups of buffaloes. This study was supported by National Research Council, Thailand (year 2002). AP is PhD candidate under Royal Golden Jubilee program, Thailand Research Fund.

The Effect of Pretreatment with Different Doses of GnRH to Synchronize Follicular Wave on Superstimulation of Follicular Growth in Dairy Cattle

The objective of this study was to evaluate the efficiency of gonadotropin releasing hormone (GnRH) and GnRH doses in synchronizing follicular wave emergence as a pretreatment for superovulation in cattle. Fourteen Holstein-Friesian cows 6 days from estrus were randomly assigned to receive 100 microg (n=4), 50 microg (n=5), or 25 microg (n=5) of GnRH. Superovulation was induced with injections of porcine FSH (pFSH) twice daily, decreasing the dose (total 42 AU) over 5 days beginning 2.5 days after receiving GnRH. On the 7th and 8th injections of pFSH, 750 microg of PGF(2alpha) was also given. With the exception of one cow that was given 50 microg of GnRH, ovulation was induced in all cows from the three groups and the new follicular wave emergence was observed. The total number of follicles for the 25 microg GnRH group was less than that observed for the 100 microg GnRH group (P<0.05), although there were no differences between the 100 microg, 50 microg and 25 microg GnRH groups with respect to the number of preovulatory follicles (>or=10 mm) and CL. The numbers of normal embryos were greater for the 25 microg GnRH group than the 100 or 50 microg GnRH groups (P<0.01); however, the numbers of ova/embryos did not differ significantly between the three groups. These results suggest that 25 microg of GnRH was sufficient to induce ovulation and follicular wave emergence. On day 6 of the estrous cycle, a reduction of the dose of GnRH to synchronize follicular wave emergence as a pretreatment for superstimulation promotes transferable embryos.

In Vivo Evidence that Local Cortisol Production Increases in the Preovulatory Follicle of the Cow

The aim of the present in vivo study was to monitor real-time fluctuations of cortisol (Cr) in the wall of preovulatory follicles using a microdialysis system (MDS) implanted in the theca layer as well as changes in ovarian venous plasma (OVP) and jugular venous plasma (JVP). Seven cows were superovulated using FSH and prostaglandin F2alpha injections. Dialysis capillary membranes were surgically implanted into the theca layer of mature follicles and connected to a microdialysis system. Fractions of the perfusates were collected from Day -1 (Day 0=LH surge) to Day 3. No difference in the concentrations of Cr between JVP and OVP was detected throughout the experiment. Circulating concentrations of Cr ranged from 20 to 35 ng/ml 8 h after surgery in ovulatory and anovulatory cows. In five ovulatory cows, the Cr concentration decreased to basal levels (<10 ng/ml) between 12 and 24 h after surgery, however, two anovulatory cows retained high Cr levels (>10 ng/ml) up to 42 h after surgery. There was a clear increase in the local concentration of Cr from 13.3+/-2.1 pg/ml at -24 h to 27.5+/-1.7 pg/ml at 0 h (peak of the LH surge) within the wall of ovulatory follicles. This increase was not detected in anovulatory follicles. This transient increase in Cr occurred only in the follicle wall, but not in the OVP or JVP, indicating that the presence of a local regulatory mechanism for Cr production/conversion in ovulatory follicles, and this mechanism may modulate the inflammatory-like reaction induced by LH surge in the follicle wall. The present results demonstrate that the glucocorticoid environment in the follicular wall adjusts at the local level in bovine ovulatory follicles. This mechanism may protect follicles from the adverse effects of glucocorticoid, and it may prevent excess inflammatory reactions associated with ovulation by temporarily increasing local concentrations of glucocorticoid, thus forming an integral part of the regulatory mechanism in ovarian physiology.

47 CHROMATIN AND CYTOSKELETAL REORGANIZATION OF RABBIT OOCYTES AFTER CUMULUS CELL NUCLEAR TRANSFER

It has been reported that a preincubation of the reconstructed oocytes prior to activation treatment might enhance the efficiency of nuclear reprogramming after somatic cell nuclear transfer in many species. The objectives of this study were to evaluate chromatin and cytoskeletal reorganization of nuclear-transferred rabbit oocytes before activation treatment. Sexually mature New Zealand White rabbits (6-month-old) were superovulated by six consecutive injections of FSH (0.4 mg) at 12-h intervals, followed by a single hCG (200-IU) injection. Mature oocytes were collected by flushing the oviducts 14–15 h post-hCG treatment. Cumulus cells were introduced into enucleated oocytes by piezo-driven microinjection. The reconstructed oocytes were randomly allocated to two treatment groups, with or without electric pulses (EP, 2 kV cm−1), and the progressive changes of the chromatin and cytoskeleton were examined by immunocytochemical staining at 0, 2, and 4 h after EP. The oocytes (n = 59) without successful enucleation served as the control, in which 30 out of 59 oocytes were electrically pulsed. After immunocytochemical staining, the proportions of reconstructed oocytes possessing dense chromatin decreased from 0 to 4 h (40-0% vs. 82-9%, respectively) in both groups, but only the oocytes without EP showed a significant reduction (P &lt; 0.05). In contrast, the proportions of reconstructed oocytes with spread chromatin increased significantly from 0 to 4 h in both groups (20–95% vs. 5–82%, respectively; P &lt; 0.05). The oocytes at 0 h after EP had the greatest percentage (35%) of normal chromosome alignment on the spindle compared to the other EP groups. The EP treatment also caused significant increase of the cytasters in both the reconstructed (5 to 45%, P &lt; 0.05) and the control oocytes (20 to 78%) at 0 h. These changes were recovered at 2 h after EP, but the proportions of cytasters in the reconstructed oocytes increased to 59% in the non-EP group and 81% in EP group at 4 h after EP. The percentages of reconstructed oocytes with multiple spindles increased from 0 to 27% and 43% in EP and non-EP groups, respectively. These results demonstrated that EP changed the proportions of chromatin configurations from the condensed form to normal alignment on the metaphase plate. The extended preincubation period for the reconstructed oocytes prior to activation treatment might increase the incidences of chromatin and spindle abnormalities of the reconstructed rabbit embryos.

191 TIMING OF OVULATION IN THE GONADOTROPHIN-STIMULATED SOUTHERN HAIRY-NOSED WOMBAT, LASIORHINUS LATIFRONS

The southern hairy-nosed wombat (SHW), Lasiorhinus latifrons, is a model species in which to develop assisted breeding techniques for the endangered northern hairy-nosed wombat, Lasiorhinus krefftii. We recently showed that anoestrus SHW respond to eutherian gonadotrophins by production of multiple ovarian follicles, but ovulation had not occurred at the time of examination 24 h post-LH (Druery GV et al. 2003 Theriogenology 59, 391 abst). This study investigated the timing of ovulation in six anoestrus captive adult female SHW (n = 3 per group) after ovarian superstimulation using porcine FSH (200 mg total, Folltropin-V, Bioniche, Belleville, Ontario, Canada) administered s.c. at 12-h intervals over 7 days. Ovulation was triggered by a single s.c. dose of porcine LH (25 mg Lutropin-V, Bioniche) 12 h after the final FSH injection. Superstimulatory response was determined by laparoscopy immediately after the final FSH injection on Day 7 prior to LH. Group 1 was re-examined at 33, 36, and 39 h post-LH, and Group 2 at 42, 45, and 48 h post-LH, for evidence of ovulations using laparoscopy and transabdominal ultrasonography. Laparoscopy on Day 7 revealed an ovarian follicular response in all six females, which coincided with the highest levels of estradiol. The reproductive tract also responded to the treatment (swollen fimbriae and enlarged, highly vascular uteri). Multiple follicles (range 16–31) up to 11 mm in diameter were observed in five females. One female had ovulated, as determined by the presence of corpora lutea. Transabdominal ultrasonographic imaging was unable to confirm the number of follicles in stimulated ovaries. Ovulation had commenced by 36 h post-LH, with the majority occurring 39–45 h post-LH. Ovulation was recorded as having occurred if a dark red, highly vascular crater on the surface of the newly formed corpus hemorrhagicum was observed. Increased circulating levels of progesterone were confirmed 9 days after the last laparoscopies. These results have important implications for the development of assisted reproductive technologies in the SHW: (1) transabdominal ultrasound imaging is ineffective for determining ovarian activity; (2) laparoscopy is a well-tolerated, repeatable minor surgical procedure that can be used for intrauterine AI in this species in which nonsurgical AI is unlikely to succeed (Paris DBBP et al. 2003 Theriogenology 59, 401 abst); and (3) knowledge of the timing of ovulation will enable insemination of spermatozoa into the uterus prior to ovulation. Financial support was provided by Dr. M. Jacobson, and hormones were supplied by Bioniche.

The effects of previous ovarian status on ovulation rate and early embryo development in response to superovulatory FSH treatments in sheep

A total of 64 ewes was used to determine if the changes in superovulatory yields related to the ovarian status at the start of superovulatory treatment are due to differences in the population of gonadotrophin-responsive follicles, alterations in the processes of ovulation or transport of embryos from oviduct to uterus and/or developmental competence of the oocyte/embryo. Ovarian status at the start of a superovulatory FSH step-down treatment, administered coincidentally with a progestagen, was assessed by ultrasonography. On Day 4 after progestagen withdrawal, embryos were recovered from oviduct and their viability was determined by assessing development in vitro culture (IVC) until the hatched blastocyst stage. In all the ewes, the ovulation rate was related positively to the number of 2–3 mm follicles at first FSH injection ( P < 0.005). However, the total number of embryos and their viability were related to the more limited category of 3 mm follicles ( P < 0.05), whereas a higher degeneration rate was related to the number of 2 mm follicles. The presence of a corpus luteum (CL) at the start of superovulatory treatment exerted a protective effect on embryonic viability, decreasing the degeneration of embryos. On the other hand, the presence of a dominant follicle at first FSH dose affected the mean size of the pool of follicles responding to the superovulation treatment, because ovulation arose from 3 to 5 mm follicles in absence of large follicles ( P < 0.05), but from 2 to 3 mm follicles when large follicles were present ( P < 0.005), indicating atresia in medium sized follicles in the presence of a large follicle.

Pharmacodynamics of a Single Low Dose of Long-Acting Recombinant Follicle-Stimulating Hormone (FSH-Carboxy Terminal Peptide, Corifollitropin Alfa) in Women with World Health Organization Group II Anovulatory Infertility

In a double-blind, placebo-controlled, randomized study, 55 anovulatory subjects received a single s.c. injection of placebo (n = 10) or recombinant long-acting FSH [FSH-carboxy terminal peptide (CTP), ORG 36286, corifollitropin alfa; NV Organon, The Netherlands] in doses of 7.5 (n = 13), 15 (n = 10), 30 (n = 11), or 60 microg (n = 11). The injection was given 2 or 3 d after the onset of a spontaneous or progestagen-induced withdrawal bleed. After drug administration, the induced follicular response varied widely among subjects in each dose group. The percentage of subjects with a follicular response (at least one follicle > or = 10.0 mm) increased with the dose (P < 0.01) and was 10, 31, 70, 73, and 82% in the placebo and 7.5-, 15-, 30-, and 60-microg treatment groups, respectively. In responding subjects, the average maximum number of follicles was 4.0, 7.6, 13.4, and 20.0, respectively, which was reached at 6.5, 6.9, 6.6, and 8.2 d after a single dose of 7.5, 15, 30, and 60 microg FSH-CTP, respectively. The dose-response for the number of follicles was statistically significant within the dose range tested (P < 0.01). Peak serum inhibin-B levels were significantly correlated with serum estradiol (E2) levels (r = 0.84, P < 0.01), and peak concentrations of inhibin-B and E2 correlated with the number of follicles observed at the same time point (for both hormones; r = 0.47, P < 0.01). Overall per treatment group, serum E2 and inhibin B concentrations significantly increased only in the two highest FSH-CTP dose groups, reaching peak concentrations at d 3 in the 30-microg group and at d 5 in the 60-microg group. Thereafter these hormone values declined rapidly, returning to baseline within 1 wk after FSH-CTP administration. In total, nine of the 55 treated subjects (16.4%) ovulated after drug administration: one subject in the placebo group, two subjects in the 7.5-microg group, three subjects in the 15-microg group, two in the 30-microg group, and one in the 60-microg group. Three subjects had monofollicular ovulation after placebo (n = 1) and a single dose of 15 microg FSH-CTP (n = 2). In two subjects with too many preovulatory follicles, (multiple) ovulation was prevented by GnRH antagonist administration. Thus, a single low dose of long-acting FSH-CTP was able to induce one or more follicles to grow up to ovulatory sizes, but the anovulatory status was not reversed because the incidence of subsequent (mono)ovulations was low.

Intermittent administration of recombinant FSH* during the first 6 days of ovarian hyperstimulation reduces the total number FSH injections

Intermittent administration of recombinant FSH* during the first 6 days of ovarian hyperstimulation reduces the total number FSH injections

The Effect Methodology of FSH Injection on The Ovulation Rates of Dairy Cattle

Abstract dalam bentuk hard copy

Plasma inhibin A determination at start superovulatory FSH treatments is predictive for embryo outcome in goats

To test whether inhibin A assays can be used for the prediction of yields in embryo programmes in goats, 50 does were treated with 45 mg FGA sponges (Chronogest ®) for 16 days plus a single dose of 100 μg i.m. cloprostenol on Day 14, just before the start of administration of eight doses of 1.25 ml of Ovagen™ twice daily for 4 days. At first FSH injection, the number and size of all follicles ≥2 mm was assessed by transrectal ultrasound and plasma inhibin A levels were measured by specific dimeric assay. There was a positive correlation between number of follicles ≥6 mm (8.8±0.5) and inhibin A levels at first FSH dose (193.2±14.5 pg/ml, P<0.05). The mean number of corpora lutea on Day 7 after sponge removal was related to the total number of follicles with a diameter of 2–6 mm at the onset of the FSH treatment (15.3±0.7, P<0.05). The total number of embryos recovered was related to the number of follicles with 4–6 mm in size (6.2±0.5, P<0.05) and to the inhibin A levels at first FSH dose ( P<0.05). These results suggest that follicles ≥4 mm are the source of inhibin prior to FSH stimulation and are the main source of oocytes resulting in the number of viable embryos recovered after a superovulatory treatment. Hence, the response to superovulatory treatments in goats in terms of the number of embryos can be predicted from either the population of follicles determined by ultrasound or the plasma inhibin A levels at start of the superovulatory FSH treatment.

Surgical Oocyte Retrieval and the Developmental Potential of the Oocytes Derived from Prepubertal Calves

The objectives of this study were to investigate the ovarian responsiveness of juvenile calves to exogenous gonadotropin treatments and to establish the oocyte retrieval technique for prepubertal heifers. Three 78-day-old calves were treated with 4 doses (40, 30, 30 and 30 mg) of FSH (Folltropin V) at 12 h interval up to 229 day-old. Surgical oocyte retrieval was performed 24 h after the last injection of FSH. Calves with good ovarian responses to FSH treatment had an average ovarian size of 5×3 cm compared to 3×2 cm in the less-responsive animals. Large variations were observed in the number of total follicles (51±45), aspirated follicles (39±36), oocytes recovered (23±25) and usable oocytes recovered (11±19) during 78 to 229 day-old. Oocytes derived from prepubertal calves had significantly lower maturation rate than those from cows (34 vs. 100%, p<0.05). Mean diameters of calf oocytes (144±1 µm) and ooplasm (110±1 µm) were significantly lower than those of cows (149±1 and 125±1 µm, respectively). The diameter of the ooplasm also increased significantly after in vitro maturation (IVM) (108±1 vs. 112±1 µm). However, further studies are required to optimize the IVP system for the oocytes derived from prepubertal heifers. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 174-178)

Human chorionic gonadotropin administration is associated with high pregnancy rates during ovarian stimulation and timed intercourse or intrauterine insemination

BackgroundThere are different factors that influence treatment outcome after ovarian stimulation and timed-intercourse or intrauterine insemination (IUI). After patient age, it has been suggested that timing of insemination in relation to ovulation is probably the most important variable affecting the success of treatment. The objective of this study is to study the value of human chorionic gonadotropin (hCG) administration and occurrence of luteinizing hormone (LH) surge in timing insemination on the treatment outcome after follicular monitoring with timed-intercourse or intrauterine insemination, with or without ovarian stimulation.MethodsRetrospective analysis of 2000 consecutive completed treatment cycles (637 timed-intercourse and 1363 intrauterine insemination cycles). Stimulation protocols included clomiphene alone or with FSH injection, letrozole (an aromatase inhibitor) alone or with FSH, and FSH alone. LH-surge was defined as an increase in LH level ≥200% over mean of preceding two days. When given, hCG was administered at a dose of 10,000 IU. The main outcome was clinical pregnancy rate per cycle.ResultsHigher pregnancy rates occurred in cycles in which hCG was given. Occurrence of an LH-surge was associated with a higher pregnancy rate with clomiphene treatment, but a lower pregnancy rate with FSH treatment.ConclusionshCG administration is associated with a favorable outcome during ovarian stimulation. Awaiting occurrence of LH-surge is associated with a better outcome with CC but not with FSH treatment.

337THE ONSET AND DURATION OF OVULATION IN DAIRY COWS SUPEROVULATED FOLLOWING SYNCHRONIZATION OF FOLLICLE WAVE WITH CIDR AND ESTRADIOL BENZOATE

Determining the optimal time for AI of superovulated cattle is important for yielding acceptable fertilization rates. The objectives of this study were to determine the interval to onset, and the duration of ovulation in dairy cows superstimulated with FSH following synchronization of follicular wave emergence with a CIDR and estradiol benzoate (EB). Holstein (n=8) and Jersey (n=1) cows received an intravaginal progesterone insert (CIDR-B, InterAg, Hamilton, New Zealand) combined with an injection of 2mg EB at a random stage of the estrous cycle (Day 0). Superstimulatory treatments were initiated on Day 6 with a total dose of 24 or 36mg FSH (Antrin, Denka, Kawasaki, Japan) via twice-daily i.m. injections in decreasing doses for 4 days. At 72h after the first FSH injection, 30mg PGF (Pronalgon F, Pharmacia, Tsukuba, Japan) was administered, and CIDR-B were removed at 80h. Transrectal ultrasonography of ovaries was performed at 3-h intervals from 40 to 77h after the PGF injection. The numbers of ovulations were counted as previously described (Purwantara et al. 1994 Anim. Reprod. Sci. 37, 1–5). All detected follicles were counted and classified as small, medium, or large (diameter 2–4mm, 5–9mm, or ≥10mm, respectively). The number of ovulations recorded after each scanning was defined as the change in the number of large- and medium-sized follicles observed since the previous scanning. Data were analyzed using ANOVA. Results are presented in Table 1. There were two patterns of ovulations: one was a concentrated pattern of ovulations (ovulatory period &amp;lt;10h); the other was a prolonged pattern of ovulations (ovulatory period &amp;gt;10h). In cows with a high superovulatory response (≥14 ovulations), the onset of ovulation was earlier (P&amp;lt;0.05) and the duration of the ovulatory period was longer (P&amp;lt;0.05) than in cows with lower (≤8 ovulations) responses. In conclusion, concentrated and prolonged ovulation patterns were seen in dairy cows superstimulated after synchronization of the follicle wave with a CIDR and EB. In cows with a large number of follicles, the onset of ovulation tended to be earlier and the duration of the ovulatory period longer than in cows with fewer follicles. Table 1 The onset and duration of ovulation in dairy cows superstimulated following synchronization of the follicle wave with a CIDR and EB

224FOLLICULAR GROWTH IN SHEEP SUPEROVULATED WITH FSH AFTER PRE-TREATMENT WITH GNRH ANTAGONISTS

In sheep, the injection of a single dose of 1.5mg of the GnRH antagonist Teverelix (Antarelix™, Zentaris, Frankfort, Germany) eliminates large dominant follicles and increases the number of smaller follicles (2–3mm in size) in a short period of time (Lopez-Alonso et al., 2003. Reprod. Abstr. Ser., 30:71). This treatment would be beneficial for increasing the efficiency of ovarian stimulatory protocols, since embryo output is enhanced in the presence of a high number of small follicles, coincidentally with the absence of large follicles, at starting the FSH treatment. However, possible effects of this single high dose of GnRH antagonist on the capacity of follicles to develop in response to FSH treatments has not been determined. In this study, we have characterized patterns of follicular development during a superovulatory treatment with purified ovine FSH (Ovagen™, ICPbio, Auckland, NZ) in sheep treated i.m. with 1.5mg of Antarelix (n=6) or saline (n=4) on Day 9 after the insertion of a progestagen sponge (Chronogest®, Intervet Int, Boxmeer, The Netherlands). Ewes were superovulated with eight decreasing doses (1.5×3, 1.25×2 and 1×3mL) of Ovagen™ injected twice daily from Day 12 of sponge insertion. Number and size of all &amp;gt;2-mm follicles was determined by transrectal ultrasonography just prior to every FSH injection from the first dose to the withdrawal of progestagen sponges. At the start of the gonadotrophin treatment on Day 12, ewes treated with GnRH antagonist showed a higher number of 2–3mm follicles than control ewes (16.2±3.8 v. 5.3±0.3, P&amp;lt;0.05), and a lower number of &amp;gt;4mm follicles (2.2±0.5 v. 5.0±0.6, P&amp;lt;0.01). Thereafter, administration of Ovagen™ induced a significant rise in the number of &amp;gt;4mm follicles at sponge removal in both groups (P&amp;lt;0.0005 for treated ewes and P&amp;lt;0.01 for controls). This number was higher in females treated with GnRH antagonist than in control sheep, although differences did not reach statistical significance (19.3±3.8 v. 12.7±5.4). Current data confirm that administration of a single dose of GnRH antagonist decreases presence of large follicles and increases the number of smaller follicles at the first day of FSH injection. This pre-treatment does not affect competence of follicles to grow in response to superovulatory FSH treatments. Moreover, the number of preovulatory follicles at sponge removal was higher than in untreated ewes. We conclude that the pre-treatment with a single dose of 1.5mg of Antarelix™ on Day 9 of sponge insertion, three days before starting the FSH treatment in a superovulatory protocol, might increase the ovarian response and, thereafter, the yields of follicles.

143DIETARY CARBOHYDRATES AND LIPIDS AFFECT IN VITRO EMBRYO PRODUCTION FOLLOWING OPU IN HEIFERS

The superstimulation protocol of Blondin et al. (2002; Biol. Reprod. 66, 38–43) produces cumulus-oocyte complexes (COCs) of high developmental competence for IVP. Using a similar protocol we assessed the affects of alterations in oocyte donor carbohydrate and lipid metabolism during ovarian stimulation on the production and viability of blastocysts in vitro. A 2×2 factorial experiment offered two diets: Fiber (F) and Starch (S) alone (0) or with 6%w/w (6) protected lipid (calcium soaps of fatty acids). Thirty-two heifers ranked by body condition score (scale: 1=thin, 5=obese) were allocated within score to one of the 4 treatments: F0, F6, S0 and S6. COCs were collected 5 days after estrus by OPU for lipid analysis. Ovarian stimulation (4 doses of FSH (9mg NIADDK oFSH) given 12h apart) commenced 2 days later. COCs were collected 40h after the last FSH injection. GnRH (0.012mg Buserelin) was administered i.v. 6h prior to OPU. A second period of ovarian stimulation and OPU then followed. Following IVM/IVF, zygotes were cultured in SOF with 0.3%w/v fatty acid-free BSA under oil (38.8°C, 5% CO2, 5% O2, 90% N) until Day 8 of development, when blastocysts were subjected to total cell counts and TUNEL analysis. Data were analyzed by ANOVA. Neither follicles aspirated (25.9±1.87) nor oocytes recovered (12.1±0.92) differed between treatments. Total fatty acids in plasma were greater (P&amp;lt;0.001) for the F than for the S diets and increased with the inclusion of protected lipid (0.75, 1.82, 0.50 and 1.39μgmL−1 for F0, F6, S0 and S6, respectively; SED=0.076). The dietary lipid-induced increase in plasma fatty acids was reflected in an increase (P&amp;lt;0.05) in total fatty acids within the oocyte (70.4, 74.7, 69.9 and 78.4 ng/oocyte; SED=3.41). Retrospective analysis by body condition indicated that S diets reduced (P=0.006) blastocyst yields in thin heifers and reduced (P=0.02) cleavage rates in fat heifers (Table 1). Blastocyst yields were lower (P=0.1) for fat heifers on the F0 diet. Total cell numbers were greater for thin heifers on S0 than F0 diet. TUNEL-positive cells averaged 4.2±0.48% and did not differ between treatments. In conclusion, modification of oocyte donor carbohydrate and lipid metabolism prior to OPU can influence IVP outcome in a complex manner dependent on body composition. Supported by Defra and The Perry Foundation. Table 1 In vitro blastocyst yields and total cell numbers