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.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

FSH-stimulated follicular secretions enhance oocyte maturation in pigs

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.     

Follicular fluid steroid levels in relation to oocyte maturity and in vitro fertilization

Steroid levels in follicular fluid (FF) obtained from stimulated ovaries in patients undergoing in vitro fertilization (IVF) were measured by capillary gas chromatography. The correlation between these levels and the maturity of the oocyte, judged from the morphology of the oocyte corona cumulus complex (OCCC) and the fertilizability of the oocytes was analysed. Oocyte maturity was associated with higher FF levels of progesterone, 17-hydroxyprogesterone, 16 alpha-hydroxyprogesterone and 20 alpha-dihydroprogesterone. Follicular fluids containing oocytes that became fertilized had significantly higher levels of 20 alpha-dihydroprogesterone and progesterone and lower levels of androstenedione. Of all the steroids determined, 20 alpha-dihydroprogesterone provides the most significant group differences. Enhanced 20 alpha-dihydrogenation in the presence of decreased 16 alpha- and 17-hydroxylation appears to be an important characteristic of the ultimate ripening stages and early luteinization, at least in stimulated cycles.     

Prolactin concentrations in follicular fluid following ovarian hyperstimulation for in vitro fertilization

Prolactin (PRL) and sex steroid concentrations were measured in follicular fluids of women treated either with (1) clomiphene/hCG or with (2) clomiphene + hMG/hCG. Method 1 of ovarian stimulation resulted in lower follicular PRL and higher oestradiol-17 beta (E2) and progesterone (P) concentrations than method 2. There was no difference in the PRL and sex steroid concentrations of follicles with fertilized and of those yielding unfertilized ova, but in both stimulation types, follicles from which no oocytes were obtained had high PRL and low E2 and P levels. Significant positive correlations were evident for PRL and T and E2 and P, respectively, while PRL and P were negatively correlated.     

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.     

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.     

Correlation between follicular fluid content and the results of in vitro fertilization and embryo transfer. III. Proteoglycans

Using a specific proteoglycan (PG) radioimmunoassay (RIA) in which human cartilage antiserum was directed against the PG protein core, the PG content of follicular fluid (FF) obtained from 42 women undergoing in vitro fertilization (IVF) and embryo transfer (ET) was studied as a function of IVF-ET outcome. Inhibition curves of purified PG cartilage preparations were parallel to those of large and small nonstimulated follicles and follicles that had been stimulated by a luteinizing hormone-releasing hormone (LHRH) agonist, d-tryptophan-6 (Decapeptyl: D-Trp6 analogue, Beaufour Laboratories, IPSEN Biotech, Paris, France), and human menopausal gonadotropin (hMG). While FF levels of immunoreactive PG-like material (Ir-PG) did not differ according to IVF-ET outcome, highly significant negative correlations were obtained between FF 17 beta-estradiol levels and FF Ir-PG levels in oocyte groups where pregnancy was obtained, i.e., oocytes were fertilized and cleaved, and pregnancy followed either for each ET or for one of two embryos reimplanted. The correlation persisted but weakened when all groups were pooled together. No correlation was observed between FF Ir-PG and progesterone. RIA or bioassay showed a positive correlation between FF inhibin and Ir-PG for the group in which each ET led to a pregnancy. Ir-PG concentrations were significantly greater in smaller than in larger follicles collected from untreated women. Upon induction of ovulation with either pure follicle-stimulating hormone (FSH), FSH + human chorionic gonadotropin (hCG), or D-Trp6/hMG + hCG, this difference no longer appeared. These results indicate that the reduction of Ir-PG concentrations constitutes an index of follicular maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of once- versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development

Ultrasound-guided transvaginal follicular aspiration combined with in vitro maturation/in vitro fertilization (IVM/IVF) and culture was used to obtain bovine preimplantation stage embryos. Evaluated were the effects of aspiration frequency on oocyte recovery and embryo development following IVM/IVF. In Experiment 1, transvaginal follicular aspiration was performed once (n=5) or twice (n=5) weekly in multiparous Angus cows with the aid of a transvaginal sector transducer (5-MHz). In Experiment 2, aspiration was performed on Angus cows once weekly (n=6), twice weekly (n=4), or twice weekly after treatment with FSH (15 mg; n=4). Follicles (>2 mm) were punctured using a 55-cm needle (17g), and oocytes were aspirated through the needle and silastic tubing (2 m) by vacuum suction (75 mmHg). The oocytes were examined for morphology and were in vitro matured and fertilized. Following IVF, all ova were co-cultured in vitro for 7 d on Buffalo Rat liver cells. Oocyte recovery rates per asp?ration session in Experiment 1 were not different between groups aspirated once or twice weekly (6.8+/-2.0 vs 6.3+/-1.1 oocytes/session; x+/-SEM) or in Experiment 2 between groups aspirated once, twice, or twice plus FSH treatment (7.7+/-1.8 vs 9.5+/-1.1 vs 6.2+/-1.1; P>0.10). In vitro development to the blastocyst stage was not different between the once, twice or twice-weekly aspiration plus FSH treatments or control oocytes obtained from cows at slaughter (23.1 vs 26.1 vs 18.0 vs 27.9%; P>0.10). Oocytes from the twice-weekly and twice-weekly plus FSH aspiration groups generated a higher percentage of Grade-1 quality embryos than the once-weekly group (P<0.05). In commercial bovine oocyte aspiration, more transferable embryos can be generated from twice-weekly aspirations than from once-weekly aspiration.     

In vitro maturation of ovarian oocytes from unstimulated rhesus monkeys: assessment of cytoplasmic maturity by embryonic development after in vitro fertilization

One-hundred and sixty-six cumulus-enclosed oocytes, obtained from ovaries of unstimulated rhesus monkeys, were subjected to six different treatments in vitro--two types of media (simple = TALP; complex = CMRL) x three levels of gonadotropins (none, FSH, FSH + hCG)--to assess their ability to undergo maturation, fertilization, and embryo development. A summary of development in culture for all experimental treatments is as follows: 58% of oocytes underwent germinal vesicle breakdown; 37% extruded a first polar body; 17% had more than one pronucleus and/or two polar bodies after insemination (i.e., were activated/fertilized); and 12% cleaved (i.e., developed) to at least the 2-cell stage in vitro. Of 45 oocytes incubated only in medium (either simple or complex) without gonadotropins, only 3 were activated/fertilized (6.7%), and only one embryo developed to at least the 2-4-cell stage (2.2%). There were no differences between oocytes incubated with only FSH and oocytes incubated with FSH + hCG. Activation/fertilization (20.7% vs. 6.7%) and embryo development (greater than or equal to 2 cells; 15.7% vs. 2.2%) were significantly higher in treatments with than without gonadotropin supplementation. There were no statistically significant differences attributable to incubation in different media during oocyte maturation. Cumulus-enclosed oocytes recovered from unstimulated ovaries of rhesus monkeys can resume maturation during culture in vitro, as shown by their ability to be fertilized and by the cleavage in vitro of the resultant zygotes.     

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.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Effect of follicle-stimulating hormone on cyclic adenosine monophosphate level and on meiotic maturation in mouse cumulus cell-enclosed oocytes cultured in vitro

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 microgram/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 +/- 0.2 to 169 +/- 13 fmol) and in the enclosed oocyte (from 0.9 +/- 0.2 to 2.4 +/- 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 microgram/ml FSH plus 1 mM isobutyl-1-methylxanthine (1BMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.     

Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

Effect of ovarian superstimulation on COC collection and maturation in alpacas

The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

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

Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge

During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% +/- 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% +/- 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% +/- 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 +/- 9) and DHT- (36 +/- 15) but not vehicle- (4 +/- 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.