Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II, and III were given 35, 18, and 9 IU recombinant human follicle-stimulating hormone (rhFSH), respectively, twice daily for 8 d; Group IV was given 18 IU rhFSH twice daily until the appearance of maximal increase in sex skin during the breeding season; and Group V was treated identically to Group II but during the nonbreeding season. In addition, nine menarchial monkeys (Group VI) were treated identically to Group II. Menarchial monkeys yielded two- to fivefold the numbers of MII oocytes (24.1) and almost twice the development potential of in vitro-fertilized oocytes (blastocyst rate: 50.0%) compared with those of the other groups. Moreover, prepubertal monkeys in Group V had approximately double the numbers of MII oocytes and in Groups IV and V twice the development potential compared with those of Groups I and II, whereas Group III did not respond to stimulation. The most prominent sex skin swelling was in association with peak serum estradiol concentrations, and good responses to stimulation were associated with reduced body temperatures. All stimulated monkeys had normal reproductive performance at adulthood, except those in Group I. In conclusion, gonadotropin stimulation of menarchial monkeys could be appropriate for addressing the high cost and limited availability of rhesus monkeys in studying reproductive biology in primates.     

Effects of rhFSH dose on ovarian follicular response, oocyte recovery and embryo development in rhesus monkeys

The objective was to study the effects of dose of recombinant human follicular stimulating hormone (rhFSH) for ovarian stimulation in rhesus monkeys. Nineteen pubertal and 109 adult female rhesus monkeys were given 37.5, 18, or 9 IU of rhFSH twice-daily for 8 days (total of 600, 300, or 150 IU of rhFSH per cycle, respectively; designated Regimens 1, 2 and 3). Ovarian responses were assessed with ultrasonography, serum concentrations of E2 and FSH, and by in vitro developmental potential (following IVF) of retrieved oocytes. Regimen 1 had more monkeys with very large follicles (diameter>8 mm) than Regimen 2 (P<0.05), which impaired development potential. However, there were no differences between Regimens 1 and 2 in oocyte recovery, whereas Regimen 3 did not elicit superovulation. The developmental potential of embryos obtained from Regimen 2 was higher than that of Regimen 1, as determined by culture to the blastocyst stage in vitro (proportion of blastocysts relative to collected MII oocytes was 55.8% versus 36.8% in pubertal and 63.8% versus 44.2% in adult monkeys; P<0.05 for each), and the results of embryo transfer from Regimen 2 were acceptable. In conclusion, we inferred that the optimal rhFSH dose for ovarian stimulation in rhesus monkeys was a total of 300 IU; this dose should be efficacious for ovarian stimulation as the quality or recovered oocytes was higher and the risk of overstimulation was reduced.     

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations, number of oocytes retrieved, and in vitro blastocyst development following IVF. One group underwent a single stimulation regimen with 18 IU rhFSH i.m., followed by 1000 IU hCG, and serum steroid concentrations and ovarian status were determined in the following three menses. Another group was stimulated as before and then allocated into three subgroups; each subgroup was re-stimulated once at the beginning of the ensuing first, second, or third menses. In the final experiment, one group was stimulated with 37.5 IU rhFSH, whereas another group received 18 IU rhFSH. In subsequent cycles, all were re-stimulated twice with 18 IU rhFSH at time intervals of two menstrual cycles (MCs). At the first menses after stimulation, serum progesterone concentrations were significantly higher and the ovaries larger than before stimulation. Monkeys that were re-stimulated at the first menses responded poorly; at the second menses, progesterone concentrations and ovarian size recovered, but the number of oocytes retrieved from re-stimulated monkeys was still significantly reduced. However, animals that were re-stimulated in two MCs later responded well (i.e., percentage of the animals responding, oocytes recovered, and potential for fertilization and blastocyst formation). In conclusion, rhesus monkeys were likely to have similar ovarian responses to repeated stimulation with the same regimen spaced at least two MCs apart.     

Ovarian stimulation of squirrel monkeys (Saimiri boliviensis boliviensis) using pregnant mare serum gonadotropin

The application of assisted reproductive technologies (ART) to nonhuman primates has created opportunities for improving reproductive management in breeding colonies, and for creation of new animal models by genetic modification. One impediment to the application of ART in Saimiri spp. has been the lack of an effective gonadotropin preparation for ovarian stimulation. Pregnant mare serum gonadotropin (PMSG) is inexpensive and readily available, but its repeated use in rhesus monkeys has been associated with induction of a refractory state. We have compared PMSG to recombinant human follicle stimulating hormone (rhFSH) for controlled ovarian stimulation in Bolivian squirrel monkeys. Groups of mature squirrel monkeys received rhFSH (75 IU daily) or PMSG (250 IU twice daily) by subcutaneous injection for 4 d during the breeding season (November to January) or nonbreeding season (March to September). Serum estradiol (E2) was measured daily. Follicular growth was monitored by abdominal ultrasound. During the breeding season, PMSG induced a higher E2 response than did rhFSH, with mean E2 levels being significantly higher within 3 d of stimulation. Superior follicular development in PMSG animals was confirmed by abdominal ultrasonography. During the nonbreeding season, PMSG elicited a similar increase in serum E2 levels despite the fact that basal serum E2 is typically low during the nonbreeding season. Repeated use of PMSG (< or = 3 cycles of administration) produced no attenuation of the E2 response. We conclude that PMSG is highly effective for repeated cycles of controlled ovulation stimulation in the squirrel monkey.     

Dynamic changes in ovarian follicles measured by ultrasonography during gonadotropin stimulation in rhesus monkeys

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulating hormone (rhFSH) for 8 d. Animals (n = 17) with ≥5 follicles (≥3 mm) in their ovaries on Day 9 of ovarian stimulation were deemed good responders, whereas those with a lesser response were poor responders (n = 4). For these two groups, the mean (±SD) numbers of oocytes retrieved were 44.3 ± 21.4 and 11.0 ± 4.6, respectively. In retrospect, the mean diameters of the ovaries and of the largest follicles, the total number of detectable follicles (diameter >0.5 mm), and serum estradiol concentrations gradually increased during the stimulation period in the good responders but did not increase in the poor responders. Comparing good and poor responders, the number of ovarian follicles >0.5 mm already exhibited a difference (12.9 ± 6.5 vs. 2.9 ± 1.3, respectively, P < 0.05) on Day 1 of stimulation. However, for other end points, differences were not significant until at least Day 5. Moreover, good responders yielded a fivefold higher blastocyst development rate than that of poor responders (P < 0.01). In conclusion, the number of ovarian follicles detected with ultrasonography could be useful to predict the response to FSH stimulation in non-human primates.     

Superovulation in ewes by a single injection of pFSH dissolved in polyvinylpyrrolidone (PVP): effects of PVP molecular weight, concentration and schedule of treatment

Three experiments were carried out to evaluate induction in ewes of superovulation and embryo production by a single injection of a porcine pituitary extract (pFSH) dissolved in polyvinylpyrrolidone (PVP), investigating the effects of PVP molecular weight and its concentration (Experiment I), time and method of treatment (Experiments II and III). All ewes were synchronized for estrus by vaginal sponges impregnated with fluorogestone acetate (FGA; 30 mg, 9 days) plus PGF(2alpha) (Cloprostenol, 50 microg, 48h before sponge removal - s.r.), and superovulated by 250 IU pFSH. In Experiment I, 60 Gentile di Puglia ewes were subdivided into five experimental groups (n = 12): Group A, the control, received six decreasing intramuscular (i.m.) doses of pFSH, 12 h apart, beginning 48h before s.r.; Groups B and C were given 48 h before s.r. a single i.m. injection of pFSH dissolved in PVP with MW = 10,000, respectively, at concentrations of 15 and 30% w/v; Groups D and E received the same treatments as for B and C using PVP with MW = 40,000. None of the pFSH-PVP treatments were effective in inducing superovulation. In Experiment II, 22 Leccese ewes were subdivided into two groups (n = 11): Group A, control received i.m. four decreasing doses of pFSH, beginning 24 h before s.r., 12h apart; Group B was given a single i.m. injection of pFSH dissolved in PVP (MW = 40,000 at 30% w/v), 24 h before s.r. The pFSH-PVP treatment provided an ovulation rate similar to the control and tended to enhance embryo yield (4.4 versus 2.4, P>0.05). In Experiment III, 60 Leccese ewes were subdivided into six treatment groups (n = 10). Groups A and D served as controls and received i.m. 12 h apart, six doses (from 48 h before s.r.) and four doses (from 24h before s.r.) of pFSH, respectively. Groups B and C were treated by a single injection of pFSH in PVP (MW = 10,000; 30% w/v) 48 h before s.r., respectively by i.m. or subcutaneous (s.c.) administration. Groups E and F received the same treatments as for B and C 24 h before s.r. Intramuscular pFSH-PVP administration 24 h before s.r. provided an ovulation rate (8.1), mean numbers of ova recovered (5.6) and fertilized (4.2) comparable to the six or four dose treatments and significantly higher (P <0.01) compared to the pFSH-PVP treatment carried out i.m. 48 h before s.r.These results show that a single injection of pFSH dissolved in PVP at 30% w/v, performed i.m. 24 h before s.r., is able to induce a superovulatory response comparable to that following multiple injection treatment, regardless of PVP molecular weight.     

Effects of recombinant human follicle-stimulating hormone on embryo development in mice

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 x CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 +/- 5.1%) and 20 (77.3 +/- 5.6%) IU rhFSH groups compared with control embryos (96.7 +/- 1.0%). The number of inner cell mass cells was reduced (P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.     

FSH is superior to eCG for promoting ovarian response in Chinese Bamei gilts

The Bamei gilt is a Chinese native breed located in northwest China, which adapts to the extremely dry and cold environment and is distinguished for its excellent reproductive and maternal characters. To ensure sufficient numbers of embryos for transgenic and nuclear transfer research, hormonal induction of gilt estrus and superovulation may be necessary. The objective of this study was to compare the superovulation effects of equine chorionic gonadotropin (eCG, Group A) and FSH (Groups B-D) in Chinese Bamei gilts. The results show that though eCG could produce more corpora lutea (CL, 14.3) than the control (CL, 9.2), and the FSH treatments had significantly increased the number of CL compared with the eCG treatment. Within the different FSH protocols, the numbers of CL were significantly greater in Groups B (CL, 77.8) and C (CL, 66.8) than in Group D (CL, 42.7), however, ovarian cysts were observed in Groups B and C, but not in Group D. These data suggest that Group D (280 IU FSH) is a suitable protocol to facilitate the development of ovarian follicles and increase the number of useful embryos per gilt for embryos recovery. The optimal FSH protocol of superovulation in Bamei gilts appears to be: D13/100 IU, D14/80 IU, D15/60 IU, D16/40 IU plus prostaglandin (PG) 0.2mg, D17/hCG 1000 IU.     

Reduced intrafollicular androstenedione and estradiol levels in early-treated prenatally androgenized female rhesus monkeys receiving follicle-stimulating hormone therapy for in vitro fertilization

Five early-treated and four late-treated prenatally androgenized and five normal female rhesus monkeys were studied to determine whether prenatal testosterone propionate exposure beginning Gestational Days 40-44 (early-treated) or 100-115 (late-treated) affects follicular steroidogenesis during recombinant human FSH (rhFSH) treatment. All monkeys underwent rhFSH injections, without human chorionic gonadotropin administration, followed by oocyte retrieval. Serum FSH, LH, estradiol (E2), progesterone (P), 17alpha-hydroxyprogesterone (17 OHP), androstenedione (A4), testosterone, and dihydrotestosterone were measured basally during rhFSH therapy and at oocyte retrieval. Follicle fluid (FF) sex steroids, oocyte fertilization, and embryo development were analyzed. Circulating FSH, E2, 17 OHP, A4, and dihydrotestosterone levels increased similarly in all females. Serum LH levels decreased from basal levels in normal and late-treated prenatally androgenized females but were unchanged in early-treated prenatally androgenized females. Serum P levels at oocyte retrieval were comparable with those before FSH treatment in all females. All prenatally androgenized females showed reduced FF levels of A4 and E2 but not P or dihydrotestosterone. Intrafollicular T concentrations also were significantly lower in late-treated compared with early-treated prenatally androgenized females or normal females. In early-treated prenatally androgenized females, but not the other female groups, intrafollicular A4 and E2 levels were reduced in follicles containing oocytes that failed fertilization or produced zygotes with cleavage arrest before or at the five- to eight-cell embryo stage. Therefore, in monkeys receiving rhFSH therapy alone without human chorionic gonadotropin administration, early prenatal androgenization reduced FF concentrations of E2 and A4 in association with abnormal oocyte development, without having an effect on P, testosterone, or dihydrotestosterone concentrations.     

In vitro fertilization and embryo transfer in the rhesus monkey

Twenty-three rhesus monkeys were subjected to 9 days of ovarian hyperstimulation with sequential exposure to human follicle-stimulating hormone (hFSH) and then human luteinizing hormone (hLH) + hFSH. Six animals (26%) did not exhibit sustained, elevated levels of circulating estradiol, primarily due to the occurrence of a premature surge of endogenous LH (n = 4). Seventeen animals (74%) responded with supraphysiologic levels of circulating estradiol (peak value: means = 4480 pg/ml) and received human chorionic gonadotropin (hCG) on Day 10. Oocytes were collected 26 h later by aspiration of large antral follicles. Oocyte quantity (means = 18/animal) and quality (63% mature) were evaluated by in vitro fertilization (IVF), embryonic development, and embryo transfer to foster mothers. Modified conditions for the successful fertilization of oocytes used a Tyrode's augmented (TALP) medium supplemented with 0.3% bovine serum albumin (BSA). Oocytes were inseminated at the metaphase II stage with ejaculated, washed sperm (50 100 x 10(3)/ml) preexposed at ambient temperature to caffeine and dibutyryl cyclic adenosine 3'5'-monophosphate. Successful fertilization ranged from 26% to 75%. In one experiment, 5 of 11 embryos produced by IVF developed in vitro to hatched blastocysts. Embryo freezing employed a propanediol-based protocol and was applied to early cleavage-stage embryos with 100% (5 of 5) post-thaw survival. Two frozen-thawed embryos were transferred transtubally on 3 occasions into rhesus monkeys during the early luteal phase of spontaneous menstrual cycles. One pregnancy resulted, which proceeded normally to the unassisted delivery of a male offspring 170 days after the LH surge. We conclude that this sequential regimen of human gonadotropins provides a cohort of oocytes from rhesus monkeys that will complete meiotic maturation and fertilize in vitro, with embryonic development proceeding in vitro and in vivo. The production of putative antibodies to human gonadotropins, assessed by the presence of Protein A-precipitated hCG binding components in sera, limits the repeated use of monkeys in the hyperstimulation protocol. Nevertheless, this model system should facilitate further studies on oocyte maturation, fertilization, and early embryogenesis in primates.     

Superovulation induction in Japanese Black cattle by a single intramuscular injection of hMG or FSH dissolved in polyvinylpyrrolidone.

The efficacy of a single intramuscular dose of 450 or 600 international units (IU) of human menopausal gonadotropin (hMG) or 30 mg of follicle stimulating hormone (FSH), each dissolved in 30% polyvinylpyrrolidone K-30 (PVP), for superovulation treatment was compared to that of superovulation induction by administration of a total dose of 600 IU hMG given in declining doses twice daily over a 3-day period. A total of 48 Japanese Black cows were used for the investigation. Oestrus was observed within 60 h after PGF2alpha administration in all cows in the hMG groups. In the hMG group that received a single dose of 600 IU hMG (n = 12), oestrus was observed less than 36 h after treatment in one cow. In contrast, oestrus was not observed in 3 of the 12 cows (25%) in the FSH group. Neither the average number of recovered ova/embryos nor the number of transferable embryos per collection differed significantly among the hMG groups. However, the average number of transferable embryos was not significantly higher in cows treated with a single dose of 600 IU of hMG than in cows treated with a single 30 mg dose of FSH (7.5+/-4.5 vs. 2.1+/-2.8). The number of cows from which more than three excellent grade embryos were collected was highest in the group that received a single dose of 600 IU hMG (9/12, 75%) and lowest in the group that received a single 30 mg dose of FSH (2/9, 22%). The differences between groups in the percentages of cows with three or more excellent embryos between treatments were not statistically significant. The proportion of recovered ova/embryos classified as excellent was highest in the group that received 600 IU hMG in declining doses and lowest in the group that received a single 30 mg dose of FSH (55.2% vs. 30.2%; P < 0.05). The recovery rate of unfertilized ova was lowest in the group that received a single dose of 600 IU hMG and highest in the group received a single 30 mg dose of FSH (18.3% vs. 48.8%; P < 0.05). Although the differences in recovery results between the groups were not statistically significant, the recovery rates in hMG groups were higher than that the FSH group. These findings suggest that superovulation can be induced adequately in Japanese Black cows using one injection of 450 to 600 IU hMG dissolved in PVP.     

Comparison of three protocols for superovulation of brown brocket deer (Mazama gouazoubira)

This study aims to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by three different protocols designed to cause superovulation in brown brocket deer. Six female received an intravaginal device containing 0.33 g of progesterone (CIDR®) for 8 days, followed by 0.5 mg injection of estradiol benzoate at the time of insertion and 265 µg of cloprostenol at the time of removal. Afterwards, the hinds were divided into three groups (n = 2): Treatment A received injection of 600 IU eCG on Day 4 after CIDR® insertion; Treatment B received injection of 300 IU eCG at the same time; and Treatment C received injection of 250 IU FSH dissolved in PVP, also on Day 4 post‐insertion. The treatments were crossed over with 44–48 day intervals after CIDR® removal, such that all the deer were submitted to all three treatments. The mean ovulation rate (Treatment A = 3.40 ± 0.68, Treatment B = 1.40 ± 0.24, Treatment C = 0.80 ± 0.49), total ovarian stimulation (Treatment A = 4.80 ± 1.02, Treatment B = 1.80 ± 0.37, Treatment C = 1.40 ± 0.60), and mean CL diameter (Treatment A = 7.33 ± 0.76 mm, Treatment B = 3.94 ± 0.19 mm, Treatment C = 2.18 ± 0.49 mm) in Treatment A were significantly higher than the mean ovulation rates, total ovarian stimulation, and mean CL diameter in Treatments B and C. The mean fecal progesterone metabolites at the luteal phase in Treatment A (6,277.94±2,232.47 ng/g feces) was significantly different from Treatment C (1,374.82±401.77 ng/g feces). Thus, although fertility was not evaluated directly, Treatment A proved capable of induce superovulation in the species Mazama gouazoubira, presenting the greatest mean ovulation rates, with the formation of functional corpora lutea. lutea. Zoo Biol 31:642‐655, 2012. © 2011 Wiley Periodicals, Inc.     

Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare

The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mares were treated similarly but with saline instead of rhFSH. The response was monitored by daily scanning of the mares' ovaries and assay of systemic oestradiol-17beta and progesterone concentrations. When the dominant follicle(s) exceeded 35 mm, ovulation was induced with human chorionic gonadotrophin; embryos were recovered on day 7 after ovulation. After an untreated oestrous cycle to 'wash-out' the rhFSH, the groups were crossed-over and treated twice-daily with 900 i.u. rhFSH, or saline. At the onset of treatment, the largest follicle was <25 mm in all mares, and mares destined for rhFSH treatment had at least as many 10-25 mm follicles as controls. However, neither dose of rhFSH altered the number of days before the dominant follicle(s) reached 35 mm, the number of follicles of any size class (10-25, 25-35, >3 mm) at ovulation induction, the pre- or post-ovulatory oestradiol-17beta or progesterone concentrations, the number of ovulations or the embryo yield. It is concluded that rhFSH, at the doses used, is insufficient to stimulate multiple follicle development in mares.     

Optimization of superovulation induction by human menopausal gonadotropin in guinea pigs based on follicular waves and FSH-receptor homologies

The guinea pig represents an excellent animal model for the study of reproduction in humans and most domestic animals because unlike the mouse and rat, it undergoes a complete estrous cycle. In this study, we investigated the availability of ovarian oocytes during the estrous cycle, and the follicle stimulating hormone (FSH) receptor (FSH-R) homologies between guinea pigs and other species, in order to identify an effective gonadotropin and optimal time-of-application for the induction of superovulation in the guinea pig. The number of collectable ovarian oocytes showed biphasic changes with peaks at the midluteal and pre-ovulatory stages. On the other hand, the number of oocytes that matured in vitro remained constant ( approximately 10 oocytes) until day 14 post-ovulation and increased thereafter. The deduced amino acid sequence of the guinea pig FSH-R showed greater similarity to the primate FSH-R than to the rodent FSH-R, which suggests that commercially available human menopausal gonadotropin (hMG) may be a better inducer of superovulation in guinea pigs. Indeed, significantly more oocytes (5.4 +/- 1.6, range 0-17, n = 10) were obtained from hMG-treated guinea pigs at the pre-ovulatory stage than during spontaneous ovulation (3.6 +/- 0.1, n = 96; P < 0.05), whereas guinea pigs that received hMG at the midluteal stage (n = 3) did not ovulate. These results indicate that hMG is an effective, albeit stage-dependent, inducer of superovulation in the guinea pig, and that FSH-R homologies should be taken into account when choosing hormones for superovulation.     

Recombinant human gonadotropins for macaque superovulation: repeated stimulations and post-treatment pregnancies

This report summarizes data from the superovulation and ultrasound-guided follicular aspiration of 40 female rhesus monkeys (Macaca mulatta) with recombinant human gonadotropins. Of the animals treated, 12 were stimulated for only one cycle, either because of a poor response to the hormones or due to ectopic ovarian position precluding ease of access via ultrasound. The majority of animals were stimulated for a minimum of 3 cycles and 3 females continued to respond for a minimum of 8 and a maximum of 10 cycles. For those animals with repeated stimulation cycles, the number of follicles developed during each of the stimulation protocols remained relatively comparable. Of the animals mated since cessation of treatment, 70% conceived. There was no difference between the conception rate in this subset of animals and the rest of the macaque breeding colony. These data indicate that participation in these studies does not impact on the reproductive potential of female rhesus monkeys.     

孕马血清促性腺激素和氯地酚对猕猴超数排卵效果的观察

用孕马血清促性腺激素,总剂量为1550—2000单位,分4—7天处理猕猴,可促使其每侧卵巢出现滤泡超数发育,随后静脉注射入绒毛膜促性腺激素2500单位,在24小时内,猕猴即可出现超数排卵。     

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis)

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33–34 days) on days 22–24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9–10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9–10 days after menses and continued for 9–10 days. When most follicles were ≥5 mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles ≥2 mm diameter was performed 30–34 h after hCG administration.

One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21±4 and 17.2±3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3 h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa     

Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 °C, 5% CO₂, and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH + 10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH + 5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14 ± 0.074 and 0.15 ± 0.076 vs. 0.46 ± 0.078 and 0.50 ± 0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.     

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley-Liss, Inc.     

Effects of estradiol-17β and hCG supplementation on superovulatory responses and embryo quality in swamp buffalo (Bubalus bubalis) implanted with norgestomet

Twenty-four cycling swamp buffaloes with normal reproductive histories and 2–3 months postpartum were used to investigate the effect of addition of estradiol-17β and human chorionic gonadotrophin (hCG) to the superovulation regime on the level of ovarian stimulation and embryo production.The estrous cycles of buffaloes were synchronized by prostaglandin injection and then divided into two groups for superovulatory treatment. Those in Group 1 (n = 12) received a implant containing 3 mg norgestomet (Syncro-Mate-B) for 9 days (insertion day is Day 0), with 4000 IU of equine chorionic gonadotrophin (eCG) and 500 μg cloprostenol i.m. given at Day 7. Group 2 (n = 12) received the same regime as Group 1, together with 7.5 mg estradiol-17β given in three intramuscular injections on Days 3, 5 and 7 in decreasing doses (4.0, 2.5 and 1.0 mg, respectively) and 5000 I.U hCG i.v. coincidentally with the first insemination. Estrus was monitored visually and by placing treated animals with bulls. Each animal was inseminated twice with frozen sperm after standing estrus. The numbers of corpora lutea (CL) and follicles greater than 8 mm in diameter were recorded via palpation per rectum at 6 days after implant removal. Two days later 11 animals from Group 2 and two from Group 1 were slaughtered for direct observation of ovarian responses and for embryo collection.The mean number of CL were 0.91 ± 0.66 and 9.08 ± 5.0 for Groups 1 and 2, respectively. The average recovery rate based on CL counts at slaughter was 60% in Group 2. No embryos were recovered from the two animals in Group 1. Seventy-nine percent of the collected ova were fertilized and more than 60% of them had developed into hatched blastocysts. The percentages of buffalo with excellent and good estrus were 41.6 and 91.6% for Groups 1 and 2, respectively.These results showed that the supplementation of estradiol-17β and the hCG treatment significantly improved the level of ovarian stimulation in swamp buffalo.