Variables affecting the yield and developmental potential of embryos following superstimulation and in vitro fertilization in rhesus monkeys

Three follicular-stimulation protocols were compared to evaluate the yield and quality of oocytes obtained from rhesus monkeys. Five animals received a high-dose regimen of PMSG (protocol I), three received a lower-dose regimen (protocol II), and two received Clomid-Pergonal (protocol III). Oocytes were recovered at laparoscopy after HCG injection, fertilized in vitro, and cultured up to the blastocyst stage. Yields of mature oocytes were 17.2 ± 13.0 (80% of total recovered), 6.7 ± 6.6 (41%), and 4.5 ± 2.5 (90%) per stimulated cycle for protocols I, II, and III, respectively. Of mature oocytes, 72%, 45%, and 89% were fertilized for protocols I, II and III, respectively. Protocol I produced the most fertilized oocytes per stimulated cycle (11.6 ± 11.6) and the greatest E₂ production (approximately fourfold that maximally expected for an unstimulated cycle). For the combined protocol I and II results, there was a significant correlation (P ? 0.05) between mean embryo development score and E₂ production. Fertilized oocytes from protocol I yielded 7.8 ± 8.0 morulae and 6.8 ± 7.2 early zonal blastocysts per cycle. After transfer of nine singleton embryos to surrogate recipients, one live birth resulted. We conclude that our high-dose PMSG regimen offers the best means at present for obtaining susbstantial numbers of developmentally competent oocytes in rhesus monkeys and that more extensive use of rapid serum E₂ assays for monitoring both stimulated cycles and those of potential surrogate recipients could help to predict the success of in vitro fertilization and embryonic development following embryo transfer in rhesus monkeys.     

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

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.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.     

Use of purified porcine follicle-stimulating hormone for ovarian stimulation of macaque monkeys

At present, in nonhuman primates, ovarian stimulation with heterologous gonadotropin preparations is the only reliable way to produce substantial numbers of competent ova for in vitro fertilization and embryo development studies. Preparations such as equine chorionic gonadotropin (eCG) and human menopausal gonadotropins (hMG or hFSH) have been used successfully, but eCG is crude and contains variable amounts of LH activity, while hMG/hFSH is very expensive and the supply is not stable. This study examined the use of a purified porcine FSH preparation (Folltropin V) for ovarian stimulation in rhesus monkeys. Twice-daily intramuscular injections of this preparation resulted in good follicular development, and was followed by a single intramuscular injection of hCG. Ova were collected laparoscopically 30 h post hCG, fertilized in vitro and then cultured until development ceased. Stimulation of 9 monkeys with Folltropin V yielded a mean of 20 ova per animal, of which 71% reached metaphase II and were inseminated; of these, 92% were fertilized in vitro and 48% developed into blastocysts in vitro. These results are similar to those reported by us and by others using eCG, hMG or an hFSH/hMG combination for ovarian stimulation of macaque monkeys. We conclude that Folltropin V is a suitable alternative preparation for ovarian stimulation in nonhuman primates and one that also has the advantages of being readily available and much less expensive than human gonadotropin preparations.     

Failure of gonadotropins to induce in vitro maturation of mouse oocytes treated with dibutyryl cyclic AMP.

In sufficient concentration, dibutyryl cAMP (DBC) prevents the spontaneous in vitro maturation of mouse oocytes. The effects of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on this inhibition were tested in an oil-free chamber-slide culture system. Mouse oocytes devoid of cumulus cells were incubated in the presence of DBC and/or gonadotropins. Oocytes cultured with follicle cells were similarly treated. Whether follicle cells were present or absent, DBC (100 or 500 mug/ml) prevented germinal vesicle breakdown in more than 95% of the oocytes cultured. Neither LH nor FSH in a wide range of concentrations acted directly on the oocytes or indirectly through the follicle cells to initiate maturation in oocytes incubated with 100 mug DBC/ml. The combination of LH (5 mug/ml) and FSH (10 mug/ml) was also ineffective in overcoming the block induced by either 100 or 50 mug DBC/ml. Maturation of oocytes in each of the DBC-free LH or FSH treatments was comparable to that occurring in control medium which did not contain exogenous gonadotropins or DBC. It was concluded that cultured oocytes treated with DBC are not a satisfactory model for studying the steps by which gonadotropins trigger the resumption of meiosis in mammalian oocytes.     

Effect of age and breeding season on the developmental capacity of oocytes from unstimulated and follicle-stimulating hormone-stimulated rhesus monkeys

Effects of age and season on the developmental capacity of oocytes from unstimulated and FSH-stimulated rhesus monkeys were examined. Immature cumulus-oocyte complexes were matured in vitro in modified CMRL-1066 medium containing 20% bovine calf serum and subjected to in vitro fertilization followed by embryo culture. After fertilization, ova from unstimulated prepubertal monkeys displayed lower development to morula (4%) than those from unstimulated adult females (18% in breeding season and 22% in nonbreeding season). No developmental difference was found between ova from unstimulated adult monkeys in breeding and nonbreeding seasons. However, ova from FSH-primed prepubertal monkeys displayed greater development to blastocyst stage (54%) than those from adult monkeys in the breeding season (16%) and nonbreeding season (0%); and ova from FSH-primed adult females in the breeding season had significantly (P < 0.05) greater developmental competence than those obtained in the nonbreeding season (> or = morula stage, 54% vs. 3%; blastocyst stage, 16% vs. 0%). These data indicate that 1) rhesus monkey oocytes acquire developmental competence in a donor age-dependent manner, and 2) animal age and breeding season modulate the effect of FSH on oocyte developmental competence in the rhesus monkey.     

Effect of aging of recipient oocytes on the development of bovine nuclear transfer embryos in vitro

The present study was undertaken to examine the effect of the length of in vitro maturation of oocytes on the efficiency of enucleation, parthenogenetic activation and blastomere fusion by electric stimulus. In vitro development of reconstituted oocytes receiving a blastomere from 8 to 16-cell bovine embryos fertilized in vitro was investigated to assess the effect of aging of the oocytes. The proportion of oocytes with a first polar body at 22 to 24 hours after maturation was high (80%) compared with those obtained at 16 to 18, 28 to 30 or 42 to 44 hours (50 to 75%). The success rate of enucleation significantly decreased with aging (88, 85, 74 and 55%). The activation rate significantly increased with the length of maturation in vitro (P<0.01) (1 to 4, 24 to 41, 57 to 70 and 80 to 87%). The proportion of oocytes fused with a blastomere from 8- to 16-cell embryos was not dependent on the age of the oocytes (54 approximately 59%). The ability of the reconstituted oocytes to develop to the 2-cell and the 8- to 16-cell stage increased with the length of maturation of recipient oocytes. When oocytes enucleated and a blastomere at 22 to 24 hours were incubated further for 22 to 23 hours until electrofusion. The proportions of oocytes which developed to the 2-cell and the 8- to 16-cell stages (74 and 17%) were similar to those obtained at 42 to 44 hours after maturation. However, only 1 to 6% of reconstituted eggs receiving a blastomere from 8- to 16-cell embryos fertilized in vitro developed into a blastocyst in vitro.     

小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

Term development of caprine embryos derived from immature oocytes in vitro

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 mug LH + 0.5 mug FSH + 1.0 mug estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82 102 (80.4%) and 50 102 (49.0%) versus 77 126 (61.1%) and 27 126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23 27 , 85.2%) and morulae (14 27 , 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4 54 , 7.4%) following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.     

Developmental capability of rat oocytes matured in vitro in defined medium

The normality of in vitro matured oocytes was compared to that of in vivo matured (ovulated) oocytes at the following stages of development: germinal-vesicle breakdown, first polar body formation, fertilization (two polar bodies and two pronuclei with a sperm tail or first cleavage), and fetal development (day 20 fetuses). At all points, the in vitro oocytes exhibited a reduced ability, with oocytes matured cumulus-free having the poorest. The exposure of oocytes to human chorionic gonadotropin (hCG) for 2 hr before collection or during incubation improved their rates of maturation and development to day 20 fetuses but not their ability to undergo fertilization. While beneficial, the exposure to gonadotropins before or during maturation was not essential, as evidenced by the production of two day 20 fetuses matured and fertilized in vitro without any gonadotropin (luteinizing hormone or hCG) treatment in vivo or in vitro. These data demonstrate that in the population of in vitro matured oocytes there exist individuals wholly competent of complete normal development, albeit in a reduced proportion in comparison to normally matured and ovulated oocytes. That the in vitro handling, treatment, and culture of the oocytes may be responsible for some of the reduced developmental ability observed is suggested by the developmental abilities of ovulated oocytes under different conditions. Ovulated oocytes fertilized in the donor had the highest rates of development (46%), followed by those fertilized after transfer into mated recipients' oviducts (20%). The lowest rate was achieved with in vitro fertilized oocytes (7%), which represented the group subject to the greatest degree of manipulation and distinction from the normal in vivo process.     

Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen

This study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus-oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 microg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6-8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6-8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.     

In vitro fertilization of gonadotropin-stimulated leopard cat (Felis bengalensis) follicular oocytes

Based on techniques developed for the domestic cat, in vitro fertilization (IVF) studies were conducted in the taxonomically related leopard cat (Felis bengalensis). Adult females received pregnant mares' serum gonadotropin (PMSG) followed 80 or 84 h later by human chorionic gonadotropin (hCG) on two to four occasions over a 40-day to 27-month interval. Oocytes were collected laparoscopically from ovarian follicles 25-27 h after hCG and co-cultured with processed, homologous spermatozoa (37 degrees C, 5% CO2 in air, humidified atmosphere) for 30-36 h. There was no apparent ovarian refractoriness to repeated treatments with exogenous gonadotropins. Overall, the mean number of mature follicles present and the total number of oocytes and proportion of immature oocytes collected did not differ (P greater than 0.05) between the 80 h (4.9 +/- 0.9; 4.7 +/- 1.2; 14.9%, respectively) and 84 h (5.6 +/- 1.4; 5.4 +/- 1.7; 22.2%, respectively) gonadotropin interval groups. However, the proportion of mature leopard cat oocytes fertilized in vitro, as determined by embryonic cleavage, was increased (P less than 0.005) by extending the interval between PMSG and hCG from 80 (17.5%) to 84 (52.4%) h. These data 1) demonstrate that, compared to the domestic cat, the ovaries of the leopard cat are less responsive to a given PMSG/hCG treatment; 2) indicate that leopard cat follicular oocytes can be recovered readily by laparoscopy and are capable of becoming fertilized in vitro; and 3) suggest that IVF may be a viable approach for producing embryos and perhaps enhancing captive propagation of rare Felidae.     

Ovum recovery and in vitro fertilization in crab-eating monkeys (Macaca fascicularis)

A total of 301 oocytes were recovered from crab-eating monkeys and subjected to insemination in vitro resulting in two fertilized ova. Sixteen monkeys in 24 cycles received 37.5 IU of hMG daily from the second day of the menstrual cycle for 7 to 10 days. Oocytes were recovered under laparotomy at 20 to 49 hr after administration of 1,000–1,500 IU of hCG. The maturation rate of the recovered oocytes was 24.2% as judged from morphological criteria under the light microscope. With additional maturation culture, the rate increased to 36.2%. The matured oocytes were inseminated at 3 to 4 hr after aspiration using homologous spermatozoa which had been capacitated in vitro. Two oocytes were judged as being fertilized based on the presence of 3 and 5 pronuclei, respectively, when examined 12 hr after the insemination. This is the first report of in vitro fertilized ova in nonhuman primates in Japan.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Maturity at collection and the developmental potential of rhesus monkey oocytes

The purpose of this study was to evaluate the in vitro fertilizability of rhesus monkey oocytes and the developmental capacity of the resulting embryos as they relate to oocyte maturation at the time of follicular aspiration. Animals were hyperstimulated with human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH), with follicular aspiration performed 27 h after administration of an ovulatory stimulus (1000 IU human chorionic gonadotropin [hCG] or 3 x 100 micrograms gonadotropin-releasing hormone [GnRH]). In 7 animals exhibiting a continuously rising pattern of serum estradiol through Day 10 of hyperstimulation, 45 germinal vesicle-intact (GV), 106 metaphase I (MI), and 24 metaphase II (MII) oocytes were collected and cultured in vitro. Upon reaching MII, oocytes were inseminated with 5 x 10(4) motile sperm/ml. Twenty-four percent of GV oocytes cultured in vitro matured to MII with 11 inseminated and none fertilized. Seventy-three percent of MI oocytes matured to MII in vitro with 50% inseminated and 32% fertilized. Oocytes collected at MII stage and inseminated underwent fertilization at a high rate of efficiency (93%). Pronuclear to 8-cell stage embryos were frozen and, upon thawing, 67% (10/15) survived with all blastomeres intact. Frozen-thawed embryos (2- to 6-cell) were transferred to the oviducts of 4 recipients (2 embryos/recipient) during the early luteal phase (1-3 days post LH surge) of natural menstrual cycles. Three twin pregnancies resulted. Thus, a positive correlation exists between the degree of nuclear maturation of rhesus monkey oocytes at collection and their potential for fertilization in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes

This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 μM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture. Mol. Reprod. Dev. 51:339–345, 1998. © 1998 Wiley-Liss, Inc.